Method of determining the probability of a therapeutic response in cancer chemotherapy with cardiac glycoside

ABSTRACT

A prognostic assay and kit and method of use thereof are provided. The kit and assay are used to determine the likelihood of a diseased cell or tissue having a therapeutic response to treatment with a cardiac glycoside in a disease having an etiology associated with excessive cell proliferation. The kit and assay are used to determine the ratio of isoforms of the α subunit of Na, K-ATPase obtained from the diseased cell or tissue. The kit can be used to predict the therapeutic responsiveness of cancer or tumor in a subject to treatment with a cardiac glycoside. The kit and assay can be incorporated in a method of treating a disease or disorder having an etiology associated with excessive cell proliferation with a composition comprising a cardiac glycoside.

CROSS-REFERENCE TO EARLIER FILED APPLICATIONS

The present application is a continuation-in-part of and claims thebenefit of PCT Application No. PCT/US2008/082641, filed Nov. 6, 2008,which claims the benefit of U.S. Provisional Application No. 60/987,501,filed Nov. 13, 2007, the entire disclosures of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention concerns a method of determining the prognosis incancer chemotherapy with a cardiac glycoside. In particular, theinvention concerns a method for determining the probability of whetheror not an in vitro or in vivo cell disease or disorder having anetiology associated with excessive cell proliferation will betherapeutically responsive to treatment with a cardiac glycoside.

BACKGROUND OF THE INVENTION

Many diseases and disorders having an etiology associated with excessivecell proliferation are fatal. The most common of these are cancer andtumors. Noncancerous proliferative diseases can also belife-threatening, however, or lead to a diminished quality of life.These may include, for example: 1) autoimmune diseases such asantigen-induced arthritis and allergic encephalomyelitis, 2) chronicinflammatory proliferative diseases such as rheumatoid arthritis,systemic-onset juvenile chronic arthritis, osteoporosis, and psoriasis,3) proliferative diseases of the breast including fibrocystic disease,4) proliferative diseases of the prostate including benign prostatichyperplasia (BPH), 5) proliferative diseases of the eye includingproliferative diabetic retinopathy, and 6) vascular proliferativediseases including atherosclerosis and coronary stenosis. Many effortshave been made to develop curative or ameliorative therapies for thesediseases and disorders; however, no comprehensive or universallycurative therapy has been developed, even though there are numerouschemotherapeutic approaches that have been proven to be effectiveagainst various different cancers, tumors and other types ofproliferative disease.

Chemotherapeutic agents are prescribed individually or in combination byclinicians in attempts to develop regimens that are tailored toindividual patients' needs. Even so, a key hurdle toward the developmentof these tailored regimens is the unpredictability of the efficacy ofchemotherapeutic agents against specific cancer or tumor phenotypes.Clinicians are forced to deal with these deadly diseases by using hit ormiss approaches. They must rely upon a historic review of the recognizedor indicated uses of particular chemotherapeutic agents and thenspeculate or guess as to whether or not a particular singlechemotherapeutic agent or combination of chemotherapeutic agents will betherapeutically effective against the cancer or tumor the clinician isattempting to cure. Such a conventional has limited success in theclinic.

Clinicians are in need of a prognostic assay that can predict with somereasonable level of certainty whether or not a particular cancer ortumor phenotype will be therapeutically responsive to a particularsingle chemotherapeutic agent or combination of therapeutic agents. Thistype of prognostic assay is extremely useful for chemotherapeutic agentsthat have a limited use history, such as those that are just enteringthe clinical environment. It would be extremely beneficial to cliniciansto have such a prognostic assay for one or more of such chemotherapeuticagents.

Preclinical studies and retrospective examination of patient data havesuggested the potential value of cardiac glycosides, (e.g. bufalin,digoxin, digitoxin, ouabain and oleandrin), in the treatment of variouscancers including breast, lung, prostate and leukemia, for example.

One of the pharmacological mechanisms of action of cardiac glycosidesinvolves their ability to bind to the ion exchange pump, Na, K-ATPaseand to inhibit the activity of this particular enzyme. Na, K-ATPase, thetransmembrane protein that catalyzes the active transport of Na⁺ and K⁺across the plasma membrane, is a well established pharmacologic receptorfor cardiac glycosides. This enzyme hydrolyzes ATP and uses the freeenergy to drive transport of K⁺ into the cell and Na⁺ out of cells,against their electrochemical gradients (Hauptman, P. J., Garg, R., andKelly, R. A. Cardiac glycosides in the next millennium. Prog.Cardiovasc. Dis. 41: 247-254, 1999).

Na, K-ATPase is composed of two heterodimer subunits, the catalyticα-subunit and the glycosylated β-subunit. There is also a γ subunit, butit has not been studied in detail. The α-subunit has binding sites forATP, Na⁺, K⁺, and cardiac glycosides. The β-subunit functions tostabilize the catalytic α-subunit and may play a regulatory role aswell. Four different α isoforms (α1, α2, α3, α4) and three different βisoforms (β1, β2, and β3) have been identified in mammalian cells. Therelative expression of each type is markedly altered in normal anddiseased states. The expression of α isoforms is tissue-type specificand varies among rodent and human tissues (Blanco, G. and Mercer, R. W.Isozymes of the Na, K-ATPase: heterogeneity in structure, diversity infunction. Am. J. Physiol. 275 (Renal Physiol. 44): F633-F650, 1998). Analtered expression of Na, K-ATPase isoforms in human cancers such asrenal, lung, hepatocellular, and colon has also been reported incontrast to those in corresponding normal tissues (Rajasekaran, S. A.,Ball, W. J., Bander, N. H., Pardee, J. D. and Rajasekaran, A. K. Reducedexpression of beta subunit of Na/K-APTase in human clear cell renal cellcarcinoma. J. Urol. 162: 574-580, 1999; Avila, J., Lecuona, E., Morales,M., Soriano, A., Alonso, T., and Martin-Vasallo, P. Opposite expressionpattern of the human Na/K-ATPase beta-1 isoform in stomach and colonadenocarcinomas. Ann. N.Y. Acad. Sci. 834: 633-635, 1997; Espineda, C.,Seligson, D. B., Ball, W. J., Rao, J., Palotie, A., Horvath, S., Huang,Y., Shi. T and Rajasekaran, A. K. Analysis of the Na, K-ATPase α- andβ-subunit expression profiles of bladder cancer using tissuemicroarrays. Cancer 97: 1851868, 2003; Jung, M. H., Kim, S. C., Jeon, G.A., Kim, S. H., Kim, Y., Choi, K. S., Park, S. I., Joe, M. K., and Kimm,K. Identification of differentially expressed genes in normal and tumorhuman gastric tissue. Genomics 69: 281-286, 2000). Additionally, theapparent affinity of cardiac glycosides to the different α isoforms isquite different. Binding of cardiac glycosides to the α1 isoform is lessthan that which occurs with the α2 and α3 isoforms which are 250-fold orhigher more sensitive to inhibition by this type of drug (Blanco, G. andMercer, R. W. Isozymes of the Na, K-ATPase: heterogeneity in structure,diversity in function. Am. J. Physiol. 275 (Renal Physiol. 44):F633-F650, 1998). Sakai et al. (FEBS Letters 563: 151-154, 2004) reportthat expression of the α3 subunit isoform is increased in humancolorectal cancer cells as compared to normal colorectal cells.

Oleandrin and oleandrigenin inhibit proliferation of human prostatecancer cells through induction of apoptosis which is due, at least inpart, to an increase in intracellular Ca²⁺ via inhibition of Na,K-ATPase (McConkey, D. J., Lin, Y., Nutt, L. K., Ozel, H. Z., andNewman, R. A. Cardiac glycosides stimulate Ca²⁺ increases and apoptosisin androgen-independent, metastatic human prostate adenocarcinoma cells.Cancer Res. 60: 3807-3812, 2000). Oleandrin and oleandrigenin alsoinhibit export of fibroblast growth factor-2 through membraneinteraction and inhibition of Na, K-ATPase activity (Smith, J. A.,Madden, T., Vijjeswarapu, M., and Newman, R. A. Inhibition of export offibroblast growth factor-2 (EGF-1) from the prostate cancer cell linesPC3 and DU145 by Anvirzel and its cardiac glycoside component,oleandrin. Biochem. Pharmacol. 62: 469-472, 2001).

While Na, K-ATPAase subunit α1 is present in many tissues because theα₁β₁ complex is considered as ‘house-keeping’ genes, α3 is predominantlydetected in excitable tissues, renal cortex, medulla, and papilla aswell as nervous tissues

Nerium oleander is an ornamental plant widely distributed in subtropicalAsia, the southwestern United States, and the Mediterranean. Its medicaland toxicological properties have long been recognized. It has beenused, for example, in the treatment of hemorrhoids, ulcers, leprosy,snake bites, and even in the induction of abortion. Oleandrin, animportant component of oleander extract, is a potent inhibitor of humantumor cell growth (Afaq F et al. Toxicol. Appl. Pharmacol. 195:361-369,2004). Oleandrin-mediated cell death is associated with calcium influx,release of cytochrome C from mitochondria, proteolytic processes ofcaspases 8 and 3, poly(ADP-ribose) polymerase cleavage, and DNAfragmentation.

It has been demonstrated that oleandrin is the principal cytotoxiccomponent of Nerium oleander (Newman, et al., J. Herbal Pharmacotherapy,vol. 13, pp. 1-15, 2001). Oleandrin is a cardiac glycoside that isexogenous and not normally present in the body. Oleandrin inducesapoptosis in human but not in murine tumor cell lines (Pathak et al.,Anti-Cancer Drugs, vol. 11, pp. 455-463, 2000), inhibits activation ofNF-κB (Manna et al., Cancer Res., vol. 60, pp. 3838-3847, 2000), andmediates cell death in part through a calcium-mediated release ofcytochrome C (McConkey et al., Cancer Res., vol. 60, pp. 3807-3812,2000). A Phase I trial of a hot water oleander extract (i.e. Anvirzel™)has been completed recently (Mekhail et al., Am. Soc. Clin. Oncol., vol.20, p. 82b, 2001). It was concluded that oleander extracts can be safelyadministered at doses up to 1.2 ml/m²/d. No dose limiting toxicitieswere found.

Ouabain, a cardiac glycoside endogenous to the body, was reported toenhance in vitro radiosensitivity of A549 human lung adenocarcinomacells but was ineffective in modifying the radioresponse of normal humanlung fibroblasts (Lawrence, Int. J. Radiat. Oncol. Biol. Phys., vol. 15,pp. 953-958, 1988). Ouabain was subsequently shown to radiosensitizehuman tumor cells of different histology types including squamous cellcarcinoma and melanoma (Verheye-Dua et al., Strahlenther. Onkol., vol.176, pp. 186-191, 2000). The cardiac glycoside oleandrin also has theability to enhance the sensitivity of cells to the cytotoxic action ofionizing radiation (U.S. patent application Ser. No. 10/957,875 toNewman, et al. and Nasu et al., Cancer Lett. Vol 185, pp. 145-151,2002). U.S. Pregrant Patent Application Publication No. 20050112059 toNewman et al. discloses the enhancement of radiotherapy in the treatmentof cancer by administration of oleandrin.

Chen et al. (Breast Cancer Research and Treatment (2006), 96, 1-15)suggest that cardiac glycosides, such as ouabain and digitalis, might beuseful toward developing anti-breast cancer drugs as both Na⁺, K⁺-ATPaseinhibitors and ER antagonists.

Smith et al. (Biochemical Pharmacology (2000), 62, 1-4) report thatANVIRZEL, and its key cardiac glycoside component oleandrin, inhibitsthe exportation of a tumor growth factor, fibroblast growth factor-2(FGF-2), from the prostate cancer cell lines PC3 and DU145.

Newman et al. (J. Experimental Therapeutics and Oncology (2006), 5,167-181) report that incubation of human malignant melanoma BRO cellswith oleandrin results in a time-dependent formation of reactive oxygenspecies, superoxide anion radicals, that mediate mitochondrial injury,loss of cellular glutathione (GSH) pools and, ultimately, tumor celldeath.

Extraction of glycosides from plants of Nerium species has providedpharmacologically/therapeutically active ingredients from Neriumoleander. Among these are oleandrin, nerine, and other cardiac glycosidecompounds. The plant extracts are useful in the treatment ofcell-proliferative diseases in animals. Oleandrin extracts obtained byhot-water extraction of Nerium oleander, sold under the trademarkANVIRZEL™, are commercially available and contain the concentrated formor powdered form of a hot-water extract of Nerium oleander.

Huachansu is an extract obtained from toad skin and it comprisesbufadienolides, such as bufalin, a cardiac glycoside. HuaChanSu is anapproved medication for the treatment of cancer in China. It has beenused to treat various cancers, including hepatic, gastric, lung, skin,and esophageal cancers.

In view of the important utility of cardiac glycosides in treatingdiseases or disorders having an etiology associated with cellproliferation, a need remains for a method of predicting the therapeuticresponse of the disease or disorder to a cardiac glycoside. No suchmethod is disclosed in or suggested by the prior art.

SUMMARY OF THE INVENTION

The invention provides a method of predicting the efficacy of a cardiacglycoside or of a composition containing a cardiac glycoside against aparticular phenotype of a disease or disorder having an etiologyassociated with excessive cell proliferation. The inventors havediscovered that the sensitivity or therapeutic responsiveness of such adisease or disorder to treatment with a cardiac glycoside is dependentupon the ratio of α3 isoform to α1 isoform expression of Na, K-ATPasesubunits in cells or tissues having the disease or proliferativedisorder. In general, the higher the ratio of α3 isoform to α1 isoformexpression of Na, K-ATPase in cells or tissues, the more sensitive(therapeutically responsive) those cells are to cardiac glycosides. Thatis, the higher the α3 isoform (drug sensitive) to α1 (drug insensitive)isoform ratio, the more sensitive that cell or tissue will be toinhibition of proliferation by cardiac glycosides.

One aspect of the invention provides an in vitro prognostic assay usefulfor predicting the in vivo therapeutic responsiveness of a disease ordisorder, having an etiology associated with excessive cellproliferation, to treatment with a cardiac glycoside or compositioncomprising a cardiac glycoside, the assay comprising:

-   determining the ratio of α3 isoform to α1 isoform of the Na,    K-ATPase α-subunit in a sample obtained directly or indirectly from    diseased in vivo cellular tissue of a subject with a disease or    disorder having an etiology associated with excessive cell    proliferation, the sample comprising one or more isoforms of the α    subunit of Na, K-ATPase; and-   determining the probability of a therapeutic response in the subject    were the subject to be treated with a therapeutically relevant dose    of cardiac glycoside according to a prescribed dosing regimen.

In some embodiments: 1) the assay further comprises predicting that thecellular tissue will be therapeutically responsive to treatment with acardiac glycoside if the ratio is greater than or equal to at least 1;2) the assay further comprises predicting that the cellular tissue willbe at least partially therapeutically responsive to treatment with acardiac glycoside if the ratio is within the range of 0.5 to 1.0; 3) theassay further comprises predicting that the cellular tissue will besubstantially therapeutically non-responsive to treatment with a cardiacglycoside if the ratio is less than 0.3; 4) the assay also furthercomprises predicting that those tissues having a subunit ratio withinthe range of 1 to 100 will be more therapeutically responsive than thosehaving an isoform ratio less than 1; 5) the assay further comprisespredicting that those tissues with only detectable α3 isoform and nodetectable α1 isoform will be the most therapeutically responsive tocardiac glycosides; and/or 6) the assay further comprises predictingthat the cellular tissue will be therapeutically responsive to treatmentwith a cardiac glycoside if the ratio is ≧2, ≧3, ≧4, ≧5, ≧7, ≧9, ≧10,≧15, ≧20, ≧25, ≧40, ≧50, ≧75 or ≧100.

In some embodiments, the probability that there will be a therapeuticresponse is related to the ratio of α3 isoform to α1 isoform of Na,K-ATPase according to the following table:

Probability that there will be a therapeutic Ratio response in thesubject 0.3 to less than 0.5 20 to <30% 0.5 to less than 1 30 to50%   >/=1 >50% >10 >75%

In the table above, the therapeutic response can be a partial or fulltherapeutic response or delayed time to progression.

In some embodiments: 1) the step of determining comprises quantifyingthe level of expression of each the α3 subunit isoform of Na, K-ATPaseand the α1 subunit isoform of Na, K-ATPase in the in vitro sample orbiopsy sample, and calculating the ratio thereof; 2) the step ofdetermining comprises determining the amount of the α3 subunit isoformof Na, K-ATPase relative to amount of the α1 subunit isoform of Na,K-ATPase in the in vitro sample, and calculating the ratio thereof; 3)the assay further comprises conducting a statistical analysis on datafrom which the ratio is determined; 4) the sample is cellular tissue,cellular mass, cellular lysate, membrane preparations prepared fromthese, or fixed histopathology slides thereof; 5) the sample is an invitro sample; 6) the sample comprises at least two isoforms of the αsubunit of Na, K-ATPase; 7) the sample comprises at least the α1 and α3isoforms of the α subunit of Na, K-ATPase; 8) the method furthercomprises lysing or disrupting cells or tissues or biopsy samples orfixing tissue sections for histopathologic examination from diseased invivo cellular tissue to form the sample; 9) the method comprisesperforming a Western blot assay or immunohistochemical staining assay onthe sample to determine the amount and relative expression of α3 subunitisoform of Na, K-ATPase relative to the α1 subunit isoform of Na,K-ATPase in the sample, and calculating the ratio thereof; 10) themethod further comprises conducting a radiometric or densitometricanalysis of a gel in order to determine the content of α3 subunitisoform of Na, K-ATPase relative to the content of α1 subunit isoform ofNa, K-ATPase in the sample; 11) the method further comprises conductinga radiometric or densitometric analysis of a gel in order to detect thepresence of and quantify the content of α3 subunit isoform of Na,K-ATPase and of α1 subunit isoform of Na, K-ATPase in the sample; 12)comparing the content of α3 subunit isoform of Na, K-ATPase and of α1subunit isoform of Na, K-ATPase in the sample relative to the content ofα3 subunit isoform of Na, K-ATPase and/or of α1 subunit isoform of Na,K-ATPase in a positive control sample and/or a negative control sample;and/or 13) comparing the content of α3 subunit isoform of Na, K-ATPaseand of α1 subunit isoform of Na, K-ATPase in a tissue sample whereexpression of only one of the α3 and α1 subunit isoforms is known tooccur as a control.

In some embodiments: 1) the diseased cellular tissue is obtained from asubject such as a mammal; 2) the diseased cellular tissue is obtainedfrom a human, cow, dog, cat, horse, pig or other domesticated animalswhether of commercial value or not; 3) the disease or disorder having anetiology associated with excessive cell proliferation is cancer or tumoror other proliferative diseases that impact adversely on human or animalquality of life; and/or 4) the cancer or tumor is selected from thegroup consisting of colorectal cancer, head and neck cancer, adrenalcortical cancer, anal cancer, bile duct cancer, bladder cancer, bonecancer, bone metastasis, sarcomas of bone, brain cancer, breast cancer,cervical cancer, non-Hodgkin's lymphoma, rectal cancer, esophagealcancer, eye cancer, gallbladder cancer, gastrointestinal carcinoidtumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi'ssarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, livercancer, lung cancer (both non small cell and small cell carcinomas),lung carcinoid tumors, malignant mesothelioma, metastatic cancer,multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasalcancer, nasopharyngeal cancer, neuroblastoma, neoplasms of the centralnervous system, oral cavity and oropharyngeal cancer, osteosarcoma,ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer,prostate cancer, retinoblastoma, salivary gland cancer, sarcoma, skincancer, stomach cancer, testicular cancer, thymus cancer, thyroidcancer, cancer of the ureter; uterine sarcoma, vaginal cancer, vulvacancer or Wilm's tumor.

In some embodiments: 1) the method further comprises identifying asubject having a disease or disorder having an etiology associated withexcessive cell proliferation; 2) the method further comprises the stepof obtaining a sample of diseased cells from the subject; 3) the methodcomprises providing information specifying how to perform analyses forthe α3 and α1 isoforms of the α subunit of Na,K-ATPase; and/or 4) themethod comprises providing information detailing how to interpretprognostic data.

In some embodiments, proliferative diseases include but are not limitedto: 1) autoimmune diseases such as antigen-induced arthritis andallergic encephalomyelitis; 2) chronic inflammatory proliferativediseases such as rheumatoid arthritis, systemic-onset juvenile chronicarthritis, osteoporosis, and psoriasis; 3) proliferative diseases of thebreast including fibrocystic disease; 4) proliferative diseases of theprostate including benign prostatic hyperplasia (BPH); 5) proliferativediseases of the eye including proliferative diabetic retinopathy; and 6)vascular proliferative diseases including atherosclerosis and coronarystenosis. In some embodiments, two or more proliferative diseases arebeing treated simultaneously.

Cancers believed to be particularly responsive to treatment with cardiacglycosides, based on laboratory research with human tumor cell lines,include prostate cancer, lung cancer, breast cancer, bladder cancer,osteogenic sarcoma, brain cancer (glioblastoma multiforma) and coloncancer. The cancers can be of human, non-human or animal origin.

In some embodiments: 1) the cardiac glycoside is selected from the groupconsisting of oleandrin, ouabain, bufalin, digitoxin, digoxin,cinobufatalin, cinobufagin, and resibufogenin; 2) the cardiac glycosideis present in pure form whether derived through extraction of a plant oranimal source, synthesized or manufactured through chemical modification(e.g. derivatization) of an available cardiac glycoside; 3) the cardiacglycoside is present in an extract; 4) the cardiac glycoside is presentin a pharmaceutical formulation or composition; 5) the cardiac glycosidehas been obtained from an oleander plant mass; 6) the oleander plantmass comprises Nerium species, such as Nerium oleander, or of Thevetiaspecies, such as Thevetia nerifolia (otherwise known as yellowoleander); and/or 7) the cardiac glycoside extract was prepared bysupercritical fluid (SCF) extraction optionally in the presence of amodifier.

In some embodiments: 1) the SCF extract further comprises at least oneother pharmacologically active agent aside from the cardiac glycoside;2) the other active agent may contribute to the therapeutic efficacy ofthe cardiac glycoside when the extract is administered to a subject; 3)the other active agent functions additively or synergistically tocontribute to the therapeutic efficacy of the cardiac glycoside; and/or4) the extract has been obtained from toad skin or secretions derivedtherefrom.

Another aspect of the invention provides a kit suitable for use inconducting the prognostic assay of the invention. The kit comprises: a)a first primary antibody having a binding affinity for the α3 subunitisoform of Na, K-ATPase; and b) a second primary antibody having abinding affinity for the α1 subunit isoform of Na, K-ATPase. The kit canbe adapted for use in conducting a Western blot gel electrophoreticassay and/or for conducting an immunohistochemical staining assay.

The kit optionally further comprises: a) a lysis composition; b) apositive control sample comprising α3 subunit isoform of Na, K-ATPase;c) a positive control sample comprising α3 subunit isoform of Na,K-ATPase and α1 subunit isoform of Na, K-ATPase; d) a negative controlsample comprising α1 subunit isoform of Na, K-ATPase and excluding α3subunit isoform of Na, K-ATPase; e) a secondary antibody, a goatanti-mouse IgG-HRP (which may be used for example for visualization ofproteins of interest); f) gel-forming material suitable for gelelectrophoretic analysis; g) radiolabeled or colored (chromic, capableof generating a visually or instrumentally detectable signal) molecularweight markers; h) instructions for use of kit and performance of theprognostic assay; i) densitometer or radiometer; j) aqueous liquidmedium; k) gel/membrane preparation kit; l) blocking solution; m) washbuffer; n) materials comprising a Western blot analysis kit; or o) acombination thereof.

In some embodiments of the kit: a) the first primary antibody hasspecific binding affinity for the α3 subunit isoform of Na, K-ATPase; b)the second primary antibody has specific binding affinity for the α1subunit isoform of Na, K-ATPase; c) the secondary antibody is Goat αmouse IgG horse radish peroxidase or comprises other secondaryantibodies from species other than mouse raised against mouse IgG withan appropriate marker attached such as horse radish peroxidase; and/ord) the primary antibodies are monoclonal antibodies.

In some embodiments, the immunohistochemical staining assay kitcomprises: a) antigen unmasking solution (which can be high pH or citricacid based); b) buffer; c) endogenous-peroxide activity-quenchingmaterial (which can comprise hydrogen peroxide, optionally in methanol);d) anti-Na,K-ATPase α3 subunit isoform antibody and/or anti-Na,K-ATPaseα1 subunit isoform antibody; e) non-immune mouse IgG1 antibody; f)universal antibody reagent comprising a mixture of anti-rabbit IgG andanti-mouse IgG reagents; g) primary chemical stain, such asdiaminobenzidine; h) a general counter-chemical stain, such ashaematoxylin or eosin; i) specific cellular organelle stain, such asthat used for staining nuclei (e.g. ethidium bromide, bisbenzimidazol orpotassium aluminum sulphate) or mitochondria (e.g. Mito tracker red,10-nonyl acridine orange); j) materials comprising animmunohistochemical staining kit; or k) a combination of two or morethereof. Using adjacent sections of tissues or cells, staining of α1subunit isoform can be done in a manner similar to that used for thestaining of α3 subunit isoform in which the appropriate specific primaryantibodies to α1 subunit isoform and α3 subunit isoform are used. Asused herein, a specific cellular organelle stain is an agent orcombination of agents used to specifically stain a particular type oforganelle (mitochondrion, nucleus, nucleolus, golgi apparatus, vacuole,etc.) in a cell (human, non-human, or animal).

In some embodiments, the immunohistochemical staining assay comprisesthe steps of: a) providing a sample of mammalian tissue; b)immunochemically staining the α3 isoform and α1 isoforms of theα-subunit of Na, K-ATPase present in the sample; c) determining thecontent of α3 subunit isoform of Na, K-ATPase and the content of α1subunit isoform of Na, K-ATPase in the sample; and d) determining theratio of α3 isoform to α1 isoform present in the sample.

In some embodiments, the immunohistochemical staining assay comprisesthe steps of: a) providing a sample of mammalian tissue; b) performingan antigen retrieval procedure on the tissue; c) quenching endogenousperoxide activity in the tissue; d) exposing the quenched tissue toanti-Na,K-ATPase α3 subunit isoform and/or anti-Na,K-ATPase α1 subunitisoform primary antibodies; e) exposing the antibody-treated tissue tosecondary anti-rabbit IgG, anti-mouse IgG antibodies or a combinationthereof; f) exposing the IgG-treated tissue to primary stain; g)exposing the stained tissue to counter-stain to form animmunohistochemically-stained tissue; h) analyzing theimmunohistochemically-stained tissue by visual or photometric means; andi) quantifying the amount of α3 and/or α1 isoforms present in themammalian tissue Quantitation of the isoform antibody staining can bedone, for example, if the secondary antibody is biotinylated. Then aVectastatin ABC stain can be added and incubated for 30 min. Followingwashes of the stained sections, they are then incubated withdiaminobenzidine substrate to develop a suitable level of staining. Thequantitation of the stained tissues can then be performed manually bygrading the intensity of the stain or electronically using computerizedimage capture and digital scanning of fields of interest. Quantitationcan be further facilitated using digital image software that iscommercially available. In some embodiments, the assay further comprisesthe step of: j) washing the tissue resulting from step a); k) washingthe tissue resulting from step c); l) providing negative controlsections (those not having tumor or cancer) as a “no primary” control;m) exposing the negative control sections with a non-immune mouse IgG1antibody; n) washing the tissue resulting from step e); o) washing thetissue resulting from step f); and/or washing the tissue resulting fromstep g). The washing can be conducted with water, buffered water, and/orTBS (about 50 mM Tris HCl, about 300 mM NaCl, about 0.1% Tween 20, pHabout 7.6).

The positive control sample can be tissue, cellular mass, cellularlysate or membrane preparations prepared from these which can beobtained through biopsy or other means of surgical excision. Thenegative control sample can be tissue, cellular mass, or cellular lysateor membrane preparations prepared from these that are known throughprior analyses not to contain α3 isoform of the α-subunit of Na,K-ATPase. In some embodiments, the negative control consists of acellular mass of rodent (mouse or rat) tumor tissue or mouse or ratcells grown in vitro.

Analytical methods that are alternative means of determining relativeNa, K-ATPase α subunit isoform composition and ratios can be employedaccording to the invention to determine the α3 to α1 isoform ratio.These might consist, for example, of use of appropriate antibodies in anELISA (enzyme linked immunoabsorbent assay) or protein tissue or celllysate array. Alternatively, it is possible to use Northern blotanalyses and related techniques (e.g. rtPCR, real time polymerase chainreaction) for measurement of mRNA to different Na, K-ATPase α-subunitisoforms. An immunohistochemical staining assay can also be used toquantify the amounts of α3 isoform and α1 isoform of the α-subunitpresent in a sample.

Another aspect of the invention provides a method of treating, in asubject, a disease or disorder having an etiology associated withexcessive cell proliferation with a composition comprising cardiacglycoside, the method comprising:

-   determining the ratio of α3 isoform to α1 isoform of the α subunits    of Na, K-ATPase in a sample obtained directly or indirectly from    diseased in vivo cellular tissue of the subject with a disease or    disorder having an etiology associated with excessive cell    proliferation, the sample comprising one or more isoforms of the α    subunit of Na, K-ATPase; and-   if the ratio is ≧0.3, ≧0.5, ≧1, or ≧10, indicating administration to    the subject a composition comprising cardiac glycoside.

Yet another aspect of the invention provides a method of treating, in asubject, a disease or disorder having an etiology associated withexcessive cell proliferation with a composition comprising cardiacglycoside, the method comprising:

-   obtaining a sample of diseased tissue from the subject, the disease    having an etiology associated with excessive cell proliferation and    the sample comprising one or more isoforms of the α subunit of Na,    K-ATPase;-   requesting that the ratio of α3 isoform to α1 isoform of the α    subunit of Na, K-ATPase in the sample be determined; and-   indicating administration to the subject a composition comprising    cardiac glycoside if the ratio is ≧0.3, ≧0.5, ≧1, or ≧10.

Yet another aspect of the invention provides a method of determiningwhether or not a subject having a disease or disorder having an etiologyassociated with excessive cell proliferation should be treated with acardiac glycoside, the method comprising:

-   obtaining a sample of diseased tissue from the subject, the disease    having an etiology associated with excessive cell proliferation, the    sample comprising one or more isoforms of the α subunit of Na,    K-ATPase;-   determining the ratio of α3 isoform to α1 isoform of the α subunit    of Na, K-ATPase in the sample; and-   if the ratio is ≧0.3, ≧0.5, ≧1, or ≧10, indicating that the subject    should be treated with cardiac glycoside by administration of a    composition comprising cardiac glycoside to the subject according to    a prescribed dosing regimen, or-   if the ratio is <0.3, indicating that the subject should not be    treated with cardiac glycoside for treatment of the disease or    disorder having an etiology associated with excessive cell    proliferation.

Some embodiments of the invention include those wherein: 1) the subjectis prescribed and administered a therapeutically relevant dose ofcomposition comprising cardiac glycoside; 2) the subject is administeredthe composition comprising cardiac glycoside according to a prescribeddosing regimen; 3) the subject is administered a composition comprisingan extract comprising a cardiac glycoside; 4) the extract furthercomprises one or more other therapeutically effective agents; 5) thecomposition further comprises one or more other therapeuticallyeffective agents; 6) the subject is administered a hot water extract ofa plant or animal source containing cardiac glycosides 2 mg to 22.5 mgper day; or 7) a concentrated extract (e.g. supercritical CO₂ extract)of a plant or animal source of cardiac glycosides ranging from 0.6 to4.8 mg; or 8) a pure single chemical form of a cardiac glycoside rangingfrom 10 to 500 ug.

The individual steps of the methods of the invention can be conducted atseparate facilities or within the same facility.

The invention includes all combinations of the aspects, embodiments andsub-embodiments of the invention disclosed herein.

BRIEF DESCRIPTION OF THE FIGURES

The following figures form part of the present description and describeexemplary embodiments of the claimed invention. The skilled artisanwill, in light of these figures and the description herein, be able topractice the invention without undue experimentation.

FIG. 1A depicts a photograph of the relevant bands of a gelelectropherogram obtained as part of a Western blot analysis of humanand mouse cell lines for relative quantitation of the α1 and α3 isoformsof the α-subunit of Na, K-ATPase.

FIG. 1B depicts a plot of concentration of oleandrin (nM) versus percentof cell growth inhibition for human and mouse tumors.

FIG. 2 depicts a photograph of the relevant bands of gelelectropherograms obtained as part of a Western blot assay of normal andmalignant human colon cells

FIG. 3A depicts a plot of concentration of oleandrin (nM) versus percentof cell growth inhibition for various different tumor cell lines varyingin their relative expressions of the of and α3 isoforms of Na, K-ATPasesubunits.

FIG. 3B depicts a bar chart of the mean IC₅₀ of oleandrin for cellgrowth inhibition in two different groups of tumor cell lines: a firstgroup having an α3 to of subunit isoform ratio of greater than one; anda second group having an α3 to α1 subunit isoform ratio of less thanone.

FIG. 4A depicts a photograph of the relevant bands of a gelelectropherogram obtained as part of a Western blot assay ofnon-transfected and transfected human tumor cells.

FIG. 4B is a bar chart demonstrating the relative expression of the α3isoform in the non-transfected and transfected cells of FIG. 4A.

FIG. 4C depicts a plot of concentration of oleandrin (nM) versus percentof cancer cell growth inhibition for the cells of FIG. 4A.

FIG. 5A depicts a photograph of the relevant bands of a gelelectropherogram obtained as part of a Western blot assay ofnon-transfected and transfected Panc-1 cells.

FIG. 5B is a bar chart demonstrating the relative expression of the α3isoform in the non-transfected and transfected cells of FIG. 5A.

FIG. 5C depicts a plot of concentration of oleandrin (nM) versus percentof cancer cell growth inhibition for the cells of FIG. 5A.

FIGS. 6A-6F depict photographs of immunohistochemically stained normalskin cells from DI 13782.

FIGS. 7A-7H depict photographs of immunohistochemically stained melanomaskin cells: FIGS. 7A-7B depict DI 15041 cells; FIGS. 7C-7D depict DI15832 cells; FIGS. 7E-7F depict DI 15833 cells; FIGS. 7G-7H depict DI15834 cells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of predicting whether or not a subjectsuffering from a disease or disorder having an etiology associated withexcessive cell proliferation will receive a clinical benefit againstthat disease or disorder by treatment of the subject with a cardiacglycoside-containing composition. The method is used to determinewhether or not the disease or disorder in the subject will betherapeutically responsive to treatment with a cardiacglycoside-containing composition. In other words, the method is used todetermine the probability of a therapeutic response in the subjectfollowing treatment thereof with a therapeutically relevant dose (oreffective dose) of one or more cardiac glycosides according to aprescribed dosing regimen.

In brief, a sample is obtained from diseased tissue in a subject. Thesample comprises one or more isoforms of the α subunits of Na, K-ATPasefrom the diseased tissue and is assayed as described herein to determinethe relative amounts or concentrations of α3 and α1 isoforms of the αsubunit of the Na, K-ATPase. The ratio of the relative amounts orconcentrations of the subunits is then calculated. If the ratio of α3isoform to α1 isoform is greater than or equal to 0.3, the methodpredicts an increased probability that the disease or disorder will betherapeutically responsive to treatment with a cardiac glycoside. Forexample, if the ratio is from 0.3-0.45+/−0.05, there is at least a 20%or a 20% to less than 30% probability that the disease or disorder willrespond to treatment with the cardiac glycoside. If the ratio is lessthan 0.2 or less than 0.3, the method predicts a less than 20%probability that the disease or disorder will be therapeuticallyresponsive to treatment with cardiac glycoside. In general, the higherthe ratio, the greater the probability is of a therapeutic response. Forexample, if the ratio is from 0.5-0.95+/−0.05, there is at least a 30%or a 30% to 50% probability that the disease or disorder will respond totreatment with the cardiac glycoside. If the ratio is from greater thanor equal to 1+/−0.05, there is at least a 50% probability that thedisease or disorder will respond to treatment with the cardiacglycoside. If the ratio is from greater than 10+/−0.05, there is atleast a 75% probability that the disease or disorder will respond totreatment with the cardiac glycoside.

By “therapeutically responsive” is meant that a subject suffering fromthe disease or disorder will enjoy at least one of the followingclinical benefits as a result of treatment with a cardiac glycoside:amelioration of the disease or disorder, reduction in the occurrence ofsymptoms associated with the disease or disorder, partial remission ofthe disease or disorder, full remission of the disease or disorder, orincreased time to progression. In other words, the therapeutic responsecan be a full or partial therapeutic response, and the method is used todetermine the probability of a therapeutic response, regardless ofwhether it is a full or partial response.

As used herein, “time to progression” is the period, length or durationof time after a disease is diagnosed (or treated) until the diseasebegins to worsen (such as until a tumor begins or continues to grow). Itis the period of time during which the level of a disease is maintainedwithout further progression of the disease, and the period of time endswhen the disease begins to progress again. Progression of a disease isdetermined by “staging” a subject suffering from a cell proliferativedisease prior to or at initiation of therapy. For example, the size,location and number of tumors a subject has is determined prior to or atinitiation of therapy. The subject is then treated with cardiacglycoside, and the size and number of tumors are monitored periodically.At some later point in time, the size and/or number of tumors mayincrease, thus marking progression of the disease and the end of the“time to progression”. The period of time during which the disease didnot progress or during which the level or severity of the disease didnot worsen is the “time to progression”.

It should be noted that a therapeutic response can be a full or partialresponse at therapeutically relevant doses to a subject according to aprescribed dosing regimen. In other words, the level of predictedtherapeutic response is determined at a dose that would not be fatal toa subject to which the cardiac glycoside is administered. Atherapeutically relevant dose, therefore, is a therapeutic doseaccording to a prescribed dosing regimen at which a therapeutic responseof the disease or disorder to treatment with a cardiac glycoside isobserved and at which a subject can be administered the cardiacglycoside without an excessive amount of unwanted or deleterious sideeffects. A therapeutically relevant dose is non-lethal to a subject,even though it may cause some side effects in the patient. It is a doseat which the level of clinical benefit to a subject being administeredthe cardiac glycoside exceeds the level of deleterious side effectsexperienced by the subject due to administration of the cardiacglycoside. A therapeutically relevant dose will vary from subject tosubject according to a variety of established pharmacologic,pharmacodynamic and pharmacokinetic principles. However, atherapeutically relevant dose (relative, for example, to oleandrin) willtypically not exceed 10, 25, 100, 250, 500 or 1000 micrograms of cardiacglycoside/day or it can be in the range of 10-500, 25-500, 10-1000 or25-1000 micrograms of cardiac glycoside/day or 2 mg to 22.5 mg ofcardiac glycoside per day. Therefore, the method of the invention isused to predict therapeutic responsiveness of the disease or disorderwhen a subject is administered a therapeutically relevant dose accordingto a prescribed dosing regimen. It is known in the art that the actualamount of a drug required to provide a target therapeutic result in asubject may vary from subject to subject according to the basicprinciples of pharmacy.

A therapeutically relevant dose can be administered according to anydosing regimen typically used in the treatment of diseases or disordershaving an etiology associated with excessive cell proliferation. Thetherapeutically relevant dose is administered according to a prescribeddosing regimen, which can be modified as need according to a subject'sclinical response. A therapeutically relevant dose can be administeredonce, twice, thrice or more daily. It can be administered every otherday, every third day, every fourth day, semiweekly, weekly, biweekly,every three weeks, every four weeks, monthly, bimonthly, semimonthly,every three months, every four months, semiannually, annually, oraccording to a combination of any of the above. For example, atherapeutically relevant dose can be administered once daily for one ormore weeks. A prescribed dosing regimen is a dosing regimen prescribedby a caregiver, such as a clinician, specifying the frequency ofadministration of therapeutically relevant doses throughout a treatmentperiod and specifying the duration of such treatment period. Forexample, a prescribed dosing regimen can include repeated administrationof one or more predetermined therapeutically relevant doses at specifiedtime intervals for a specified treatment period. The therapeuticallyrelevant dose, predetermined time interval and treatment period can bechanged dependently or independently of one another as needed and asdetermined by a caregiver.

By “lysis composition” is meant one or more agents capable of lysing themembrane of a cell to form a cell lysate or complete dissolution ofcellular contents. The lysis composition can comprise one or more of thefollowing: aqueous liquid medium, buffering agent, salt, chelatingagent, surfactant, anti-foaming agent, protease inhibitor, andphosphatase and ATPase inhibitor.

As used herein in relation to the kit, a “positive control sample”comprises a quantifiable amount or concentration of α3 subunit isoformof Na, K-ATPase. The positive control can further comprise aquantifiable amount or concentration of α1 subunit isoform of Na,K-ATPase. The quantifiable amounts or concentrations of each subunitisoform independently can be predetermined or known. The amount orconcentration of subunit isoform will be sufficient to provide apositive response for presence of the subunit isoform as determinedaccording to the particular method used in an assay for which it is apositive control. For example, in a Western blot assay, the amount orconcentration of subunit isoform in the positive control will besufficient to bind with a corresponding antibody such that when thesubunit presence is determined, for example by densitometric orradiometric determination, a positive response for the presence of thesubunit isoform will be obtained. For the immunohistochemical stainingassay (such as detailed in Example 27), the amount or concentration ofsubunit isoform in the positive control will be sufficient to permitfacile visualization and quantitation when applied to a gel, separatedfrom other proteins using electrophoresis and then stained with the useof primary and secondary antibodies as previously described or whenapplied to quantitative analysis under microscopic examination usingdigital image capture and quantitative image analysis software. If theamount or concentration of subunit isoform in the control sample isknown or predetermined prior to analysis, the level of positive responsecan be related to the amount or concentration so as to provide acalibration curve for the subunit isoform.

If α3 subunit isoform of Na, K-ATPase is present in the positive controlsample, the ratio of α3 isoform to α1 isoform will generally be withinthe range of 0.3 to 100 or greater. Suitable ranges for the ratio alsoinclude 0.3 to 0.5, 0.5 to 1.0, 1.0 to 20, and 20 to 100.

As used herein in relation to the kit, a “negative control sample”comprises an amount or concentration of α1 subunit isoform of Na,K-ATPase but excludes the α3 subunit isoform of Na, K-ATPase. The amountor concentration of α1 subunit isoform will be sufficient to provide apositive response for presence of the isoform as determined according tothe particular method used in an assay, such as the Western blot assay,in which it is used as a negative control. The amount or concentrationcan be known or predetermined and, if so, can be used to develop acalibration curve for the isoform.

As part of a validation of the method and kit of the invention, theinventors conducted preclinical evaluations of a cardiacglycoside-containing composition for the treatment of various cancer andtumor phenotypes. The evaluation was conducted using in vivo, ex-vivo,in vitro and in xenograft animal models. For each phenotype evaluated, acellular sample of tissue or cell mass was analyzed as described hereinto determine the ratio of α3 subunit isoform of Na, K-ATPase to α1subunit isoform of Na, K-ATPase. A correlation between the phenotypesthat are responsive to cardiac glycoside treatment and the ratio of α3subunit isoform of Na, K-ATPase to α1 subunit isoform of Na, K-ATPasefor each of the phenotypes in a number of human tumor cell lines wasobserved. It was concluded that a therapeutically responsive cancer ortumor phenotype (or one with an increased probability of a therapeuticresponse) possesses a ratio of α3 subunit isoform of Na, K-ATPase to α1subunit isoform of Na, K-ATPase of at least 0.3.

Rodent (rat or mouse) cancer cells can be used as a negative controlsample. FIG. 1A depicts a film developed against the gel of a Westernblot analysis of rodent tumor cell lines. The inventors' datademonstrate that all rodent (mouse and rat) tumor cell lines tested todate lack the α3 subunit isoform and that their proliferation in vitrois not significantly inhibited by incubation with cardiac glycosides(e.g. ouabain, oleandrin, bufalin, etc.). Human tumor cell lines aregenerally more sensitive to treatment with cardiac glycoside-containingcomposition than are murine tumor cells (FIG. 1B). The inventors believethis difference in therapeutic responsiveness is due to the differencesin the ratio of α3 to α1 isoforms in the two different species, as notedabove.

It should be noted that the antibodies against α3 isoform can be derivedfrom a number of different vendors including Affinity Bioreagents(Golden, Colo.), Novus (Littleton, Colo.) or Sigma hemical Co. (StLouis, Mo.) all of which showed equal reactivity (i.e. binding) to theα3 epitope (isoforms) in a positive control sample.

In humans, the α3 to α1 isoform ratio for the α subunit of Na, K-ATPasemay vary according to the source and state of malignancy of the tissue.Tissue samples from normal and malignant colon tissue were taken fromeach subject for a total of fourteen individual subjects. The α3 to α1isoform ratio for each tissue sample was determined as described herein.The data in FIG. 2 and the table below demonstrate a relative lack of α3isoform in normal tissues, and the presence of α3 isoform inapproximately 66-70% of malignant tissues. Both normal and malignanttissues contain the α1 subunit isoform.

Normal colon tissues Colon tumor tissues Subject α3/α1 ratio α3/α1 ratio1 0.0 0.0 2 0.02 0.64 3 0.03 0.07 4 0.01 0.04 5 0.03 0.0 6 0.0 0.56 70.0 3.4 8 0.0 6.0 9 0.0 100.0 10 0.0 0.0 11 0.0 6.2 12 0.0 0.83 13 0.00.36 14 0.0 0.00

The α3 to α1 isoform ratio for the α subunit of Na, K-ATPase wasdetermined for various different human cancer and tumor phenotypes incultured cell lines as well as actual tumor biopsy samples. In addition,the relative sensitivity of each phenotype to treatment with a cardiacglycoside-containing composition was determined using data derived fromin vitro cell culture experiments. The data for an oleandrin-containingcomposition are detailed in the following table and FIG. 3A.

In vitro response Cell line Tumor type α3/α1 to oleandrin? IC₅₀ (nM)MDA231 Breast 0.01 No* N/A BXPC3 Pancreas 0.29 No* N/A MCF7 Breast 0.2No* N/A SUM149 Breast 0.3 Partial** 22.5 HCT116 Colon 1.7 Partial** 36.2MiaPaca Pancreas 1.5 Complete*** 14.2 CACO2 Colon 4.6 Complete*** 9.4HT29 Colon 6.8 Complete*** 15.6 LS174T Colon 7.6 Complete*** 20.7 BROMelanoma 11.5 Complete*** 8.2 DOD-1 Colon 19.8 Complete*** 19.6 PANC1Pancreas 60 Complete*** 6.1 In vitro response Cell line Tumor type α3/α1to bufalin? IC₅₀ (nM) BXPC3 Pancreas 0.29 No* N/A MiaPaca Pancreas 1.5Complete*** 2.1 PANC1 Pancreas 60 Complete*** 1.2 *Denotes less than 50%growth inhibition achieved at concentrations up to 125 nM **Denotesgreater than 50% but less than 75% growth inhibition achieved at 62 nM***Denotes greater than 75% growth inhibition achieved at 62 nM

As defined in the Tables above, an “in vitro response” denotes theextent to which proliferation of a given tumor cell line is inhibitedwith a stated concentration of drug (cardiac glycoside) over a definedperiod of time. A designation of “no response” denotes less than 50%growth inhibition at any of the multiple drug concentrations tested.That is, no concentration could be identified as an IC₅₀ value (thatconcentration producing inhibition of cell growth by 50% over the statedperiod of time of the experiment). Accordingly, were this tumor cellline growing in a mammal such as a human, it is probable that the drugwould be ineffective in producing significant inhibition of tumorgrowth. The designation of “partial response” in the Table above denotesthe capacity of a given cardiac glycoside such as oleandrin or bufalinto inhibit cell proliferation by more than 50% but less than 75%relative to nontreated tumor cell growth. Accordingly, this would equateto a partial or less than complete therapeutic response of the tumor tothe cardiac glycoside if this product were used to treat tumor growth ina mammal such as a human. Finally, the designation of “completeresponse” in the Table above denotes greater than a 75% inhibition oftumor cell proliferation relative to untreated tumor cell populations.Accordingly, it is probable that total inhibition of tumor growth (or acomplete therapeutic response, in a mammal might be achieved.

Based upon the isoform ratio data above, the method predicts: 1) MDA231,BXPC3 and MCF7 cell lines would be substantially non-responsive totreatment with a therapeutically relevant amount of cardiac glycoside;2) SUM149 and HCT116 cell lines would exhibit at least a partialresponse to treatment with a therapeutically relevant amount of cardiacglycoside; and 3) CACO2, HT29, LS174T, BRO, DOD-1 and PANC1 cell lineswould exhibit a full therapeutic response to treatment with atherapeutically relevant amount of cardiac glycoside.

FIG. 3A depicts a plot of concentration (nM) of oleandrin (provided asoleandrin) versus the percentage of growth of control. The datademonstrate that the cell lines evaluated can be categorized into threedifferent groups according to their response to cardiac glycosidetreatment as determined by relative inhibition of proliferation of thehuman tumor cell lines: Group I—cells that undergo less than 50% growthinhibition achieved at concentrations up to 125 nM of cardiac glycoside;Group II—cells that undergo greater than or equal to 50% growthinhibition achieved at concentrations up to 62 nM of cardiac glycoside;and Group III—cells that undergo greater than or equal to 75% growthinhibition achieved at concentrations up to 62 nM of cardiac glycoside.Exemplary cell lines for the groups are: Group I—MDA231, BXPC3, MCF-7;Group II—SUM149, BRO; Group III—Panc-1, CaCo2. The response data are aspredicted by the isoform ratio data. In order to establish therobustness of the method and kit of the invention, a statisticalanalysis of cellular response data was conducted. The data in FIG. 3Bdepict the relative ability of oleandrin-containing composition toinhibit human tumor cell proliferation in vitro. The data are depictedas Mean±SD. Oleandrin mediated growth inhibition was examined in tencell lines (representing a variety of solid tumors) in which therelative subunit content α3/α1 isoform ratio >1.0 and six cell lines inwhich the α3/α1 isoform ratio <1.0. The data show that there is an8-fold (800%) difference between these groups. That is, in those celllines in which the α3/α1 isoform ratio ≧1.0, oleandrin was found to be amuch more effective drug as compared to those in which the α3 subunitisoform was only poorly expressed. The data prove that the ratio ofα3/α1 subunit isoforms can be used to predict the responsiveness of acancer or tumor cell line to treatment with a cardiacglycoside-containing composition.

Further proof of the relative importance of the α3/α1 isoform ratiotoward predictability of a therapeutic response by a target malignanttissue to treatment with a cardiac glycoside was established as follows.Artificial down-regulation of the expression of α3 isoform in humanpancreatic cancer cells, i.e. manipulation of isoform content ofsubunits of Na, K-ATPase, was achieved by transfection of cells withsiRNA (silencing RNA) specific to the α3 isoform. The data demonstrate adecline in expression of the α3 isoform (as determined by Western blot(FIG. 4A)). Quantitation of the decreased expression of α3 by Westernblot is detailed in FIG. 4B. FIG. 4C demonstrates that the transfectedcells, in which the α3 expression has been reduced, lose theirsensitivity to treatment with oleandrin.

Still further proof of the relative importance of the α3/α1 isoformratio toward predictability of a therapeutic response by a targetmalignant tissue to treatment with a cardiac glycoside was establishedby transfection of Panc-1 cells with α1 subunit isoform. The Na,K-ATPase α1 subunit isoform was transfected to Panc-1 cells, whichnormally lack α1 expression. The control sample in FIG. 5A is fornon-transfected Panc-1 cells, and the α1cDNA sample in FIG. 5A is forPanc-1 cells transfected with the α1 subunit isoform. As a result, theratio of α3 to α1 was reduced in the transfected Panc-1 cells (FIG. 5B).The sensitivity of the transfected Panc-1 cells to oleandrin treatmentwas then evaluated. The sensitivity of these transfected cells tooleandrin treatment was reduced as evidenced by the shift of IC50 valueof oleandrin from 4.1 nM of non-transfected Panc-1 cells to more than 50nM in α1 transfected cells (FIG. 5C). Accordingly, the antiproliferativeactivity of oleandrin was markedly reduced in Panc-1 cells transfectedwith the α1 subunit isoform.

Detection and quantitation of the α3 and α1 isoforms of the Na, K-ATPasesubunit can also be accomplished by immunohistochemical staining, suchas detailed in Example 27. Cells are immunohistochemically stained suchthat the α3 and α1 isoforms are stained differentially. The content ofeach isoform type is quantified and the ratio of content of the α3 to α1isoform is determined. Differential immunohistochemical staining of theα3 and α1 isoforms can be accomplished by various means. In someembodiments, a tissue sample is stained with two different stains, onestain being used selectively or specifically for the α3 isoform and theother stain being used selectively or specifically for the α1 isoform.In some embodiments, two close but different samples are obtained fromthe same tissue and stained, such that the first sample is treated tostain the α3 isoform and the second sample is treated to stain the α1isoform. The amounts of the α3 and α1 isoforms are then quantified, andthe ratio of α3 isoform to α1 isoform is then determined. Based upon theratio, a prediction is made as to the likelihood of a therapeuticresponse of the tissue to treatment with cardiac glycoside.

Quantitation of the α3 isoform and the α1 isoform of the Na, K-ATPasesubunit in an immunohistochemically stained sample (cell or tissueslide) can be accomplished through manual or automated procedures. Theseinclude but are not limited to individual grading of staining intensityof set areas of suitably stained slides (e.g. using visual observationsof relative staining intensities (e.g. intensities of 0-5 where 0 is nodetectable staining and 5 represents intense staining), then compilingaverage staining intensities of a set number of examined regions andcomparing relative staining of α3 staining to that of slides stained forα1, and calculating the ratio of α3 to α1 in the sample. Another mannerin which quantitation of the relative staining of α1 and α3 (i.e. theratio of α3 to α1 present in a sample) can be accomplished through theuse of microscopic examination of set areas of stained slides and theuse of digital analysis software to determine relative stainingintensities Likewise computer assisted digital video analysis can alsobe performed to determine relative isoform staining and to compute aratio of α3 to α1 staining intensities, thereby determining the ratio ofα3 isoform to α1 isoform content in the sample.

For example, FIGS. 6A-6F depict photographs of normal skin cells from DI13782. FIGS. 6A, 6C and 6E depict α subunit of Na⁺/K⁺ ATPaseimmunoreactivity (immunohistochemically stained). FIGS. 6B, 6D and 6Fdepict photographs of the corresponding non-immune (control) IgGincubated sections. Strong 2+ or 3+ levels of immunoreactivity were seenin all donors of normal skin. The squamous epithelium, hair follicleepithelium, basement membrane of the sebaceous glands, sweat coils andmononuclear lymphocytes in blood vessels were all positively stained.Pyloerector muscle was weakly immunoreactive.

FIGS. 7A-7H depict photographs of melanoma cells. FIGS. 7A, 7C, 7E and7G depict α subunit of Na⁺/K⁺ ATPase immunoreactivity(immunohistochemically stained). FIGS. 7B, 7D, 7F and 7H depict thecorresponding non-immune (control) IgG incubated sections. FIGS. 7A and7B depict DI 15838 cells. FIGS. 7C and 7D depict DI 15840 cells. FIGS.7E and 7F depict DI 15842 cells. FIGS. 7G and 7H depict DI 15844 cells.All BCC samples were positive for the α subunit of Na⁺/K⁺ ATPase withvarying degrees of intensity. All immunoreactivity was localized to thenuclei and cytoplasm of the tumor cells. Following differentialimmunohistochemical staining of the tissues, the ratio of α3 subunitisoform to α1 subunit isoform can be determined and a prediction as totherapeutic responsiveness to treatment with cardiac glycoside can bemade.

The relevance of the method of the instant invention toward anothercardiac glycoside was evaluated. Huachansu is a toad skin extractcontaining cardiac glycosides known as bufadienolides. The therapeuticresponsiveness of two human pancreatic cell lines (SW1990 and Panc-1) totreatment with huachansu was evaluated. Based upon the data in thetables above, one would predict that SW1990 cell would not be but thatPanc-1 cells would be therapeutically responsive to huachansu. Theresponse data provided the expected results.

The examples below include evidence of the efficacy of cardiacglycosides in treating cancer and tumor related diseases and disorders.Example 21 includes a case history for treatment of a patient presentingwith metastatic pancreatic gastrinoma. The prognostic assay, methods andkit of the invention can be used in combination with one or more otherprognostic or diagnostic assays, methods and kits known in the art ofdiseases or disorders having an etiology associated with excessive cellproliferation. For example, if a clinician intends to treat a subjecthaving cancer or tumor with a combination of a cardiac glycoside andanother chemotherapeutic agent or radiation therapy, and it is knownthat the particular phenotype of cancer or tumor, which the subject has,is at least partially therapeutically responsive to treatment with saidother chemotherapeutic agent or radiation therapy, then the presentinvention can be used to determine the probability of at least a partialtherapeutic response of the cancer or tumor in the subject when treatedwith cardiac glycoside. If the results indicate that there is anincreased probability that the cancer or tumor will be therapeuticallyresponsive to treatment with cardiac glycoside, the clinician can thenprescribe and/or administer treatment of the cancer or tumor with thecardiac glycoside and other therapeutic agent or radiation therapy or acombination thereof.

The cardiac glycoside can be any cardiac glycoside known to possesstherapeutic activity in the treatment of a disease or disorder having anetiology associated with excessive cell proliferation. The cardiacglycoside can be present in pure form or as a mixture with one or moreother compounds. The cardiac glycoside can be present as an extract. Theextract can be prepared by supercritical fluid (SCF) carbon dioxide(CO₂) extraction or a chemically modified form of such an extract (e.g.an extract that includes ethanol or was made using SCF CO₂ and ethanol).The extract can be obtained from plant or animal material. The animalmaterial can be the exudate of a toad (e.g. Bufo bufo). The plantmaterial can be plant mass such as obtained from Nerium species, such asNerium oleander, or of Thevetia species, such as Thevetia nerifolia orThevetia puruviana (otherwise known as yellow oleander). The extractionprocess can be conducted on a dried powder of Nerium oleander leavesprepared according to a process described in a currently-pending U.S.provisional application Ser. No. 60/653,210 filed Feb. 15, 2005 in thename of Addington or U.S. application Ser. No. 11/340,016 filed Jan. 26,2006 in the name of Addington, U.S. application Ser. No. 11/191,650filed Jul. 28, 2006 (now U.S. Pat. No. 7,402,325 issued Jul. 22, 2008)in the name of Addington, or PCT International Patent Application No.PCT/US06/29061 filed Jul. 26, 2006, the entire disclosures of which arehereby incorporated by reference, or by a process described herein.

As used herein, the term “oleandrin” is taken to mean all known forms ofoleandrin unless otherwise specified. Oleandrin can be present inracemic, optically pure or optically enriched form. Nerium oleanderplant material can be obtained, for example, from commercial plantsuppliers such as Aldridge Nursery, Atascosa, Tex.

The extract can be obtained by modified (e.g. ethanol) or unmodifiedsupercritical fluid extraction of a cardiac glycoside-containing plantmass. The supercritical fluid extract can comprise at least one otherpharmacologically active agent that contributes to the therapeuticefficacy of the cardiac glycoside when the extract is administered to asubject. It can contribute additively or synergistically to thetherapeutic efficacy of the cardiac glycoside.

The extract can be prepared by various different processes. The extractcan be prepared according to the process developed by Dr. Huseyin ZiyaOzel (U.S. Pat. No. 5,135,745) describes a procedure for the preparationof the extract of the plant in water. The aqueous extract reportedlycontains several polysaccharides with molecular weights varying from 2KD to 30 KD, oleandrin and oleandrigenin, odoroside and neritaloside.The polysaccharides reportedly include acidic homopolygalacturonans orarabinogalaturonans. U.S. Pat. No. 5,869,060 to Selvaraj et al.discloses hot water extracts of Nerium species and methods of productionthereof. The resultant extract can then be lyophilized to produce apowder. U.S. Pat. No. 6,565,897 (U.S. Pregrant Publication No.20020114852 and PCT International Publication No. WO 2000/016793 toSelvaraj et al.) discloses a hot-water extraction process for thepreparation of a substantially sterile extract. Erdemoglu et al. (J.Ethnopharmacol. (2003) November 89(1), 123-129) discloses results forthe comparison of aqueous and ethanolic extracts of plants, includingNerium oleander, based upon their anti-nociceptive and anti-inflammatoryactivities. Organic solvent extracts of Nerium oleander are disclosed byAdome et al. (Afr. Health Sci. (2003) August 3(2), 77-86; ethanolicextract), el-Shazly et al. (J. Egypt Soc. Parasitol. (1996), August26(2), 461-473; ethanolic extract), Begum et al. (Phytochemistry (1999)February 50(3), 435-438; methanolic extract), Zia et al. (J.Ethnolpharmacol. (1995) November 49(1), 33-39; methanolic extract), andVlasenko et al. (Farmatsiia. (1972) September-October 21(5), 46-47;alcoholic extract). U.S. Pregrant Patent Application Publication No.20040247660 to Singh et al. discloses the preparation of a proteinstabilized liposomal formulation of oleandrin for use in the treatmentof cancer. U.S. Pregrant Patent Application Publication No. 20050026849to Singh et al. discloses a water soluble formulation of oleandrincontaining a cyclodextrin. U.S. Pregrant Patent Application PublicationNo. 20040082521 to Singh et al. discloses the preparation of proteinstabilized nanoparticle formulations of oleandrin from the hot-waterextract.

The SCF extraction can be conducted in the presence of a modifier in thesupercritical fluid, such as ethanol, to enhance extraction of thedesired compound(s) from the plant mass. Modifiers generally possessvolatility between that of the supercritical fluid and of the compoundbeing extracted, and they must be miscible with the supercritical fluid.In some embodiments, the modifier is a liquid at ambient conditions. Byway of example and without limitation, a modifier can be selected fromthe group consisting of ethanol, methanol, propanol, acetone, ethylacetate, methylene chloride, etc.

The extract is a mixture of pharmacologically active compounds, such asoleandrin or other cardiac glycosides, oleaside, and other plantmaterials. Oleandrin extract from a supercritical fluid process containsby weight a theoretical range of 0.9% to 2.5% oleandrin. SCF extractscomprising varying amount of oleandrin have been obtained. In oneembodiment, the SCF extract comprises about 2% by wt. of oleandrin.

As evidenced by the data herein, the SCF extract comprises a mixture ofvarious components. Some of those components include oleandrin, oleasideA, oleandrigenin, neritaloside, odorside (Wang X, Plomley J B, Newman RA and Cisneros A. LC/MS/MS analyses of an oleander extract for cancertreatment, Analytical Chem. 72: 3547-3552, 2000), and other unidentifiedcomponents. The SCF extractable unidentified components of the SCFextract appear to include at least one other pharmacologically activecomponent that contributes to the efficacy of the oleandrin in the SCFextract. That is, at least one other SCF extractable component functionsadditively or synergistically with the oleandrin to provide the observedefficacy.

It is possible that the extracts also differ in their relativeperformance as determined by efficacy against several tumor cell lines.Even so, if a cardiac glycoside is present in a sufficiently high amountor concentration in the extract to be able to prepare a therapeuticallyrelevant dose, then the extract is considered part of the invention. Thetable below summarizes some of the relative efficacy data for threedifferent forms of the cardiac glycoside oleandrin.

Human melanoma BRO cells Human pancreatic cancer DRUG (IC₅₀, μM) PANC-1cells (IC₅₀, μM) Pure Oleandrin 0.017* 0.01 Hot water extract 0.052 0.03Comprising oleandrin and complex polysaccharides Supercritical CO₂extract 0.007 0.004 comprising oleandrin and related cardiac glycosides*The IC₅₀ of tested compounds are presented as micromolar (μM) oleandrinequivalent concentrations in those extracts. That is, the data representthat concentration of oleandrin as free chemical or as part of anextract necessary to inhibit growth or proliferation of tumor cellgrowth compared to untreated cells by 50%.

As shown in the table above, the IC₅₀ value of the supercritical CO₂extract is only 50% of that oleandrin alone in both Panc-1 and BROcells, which suggests that the supercritical CO₂ extract of oleander isat least two-fold stronger (more potent) than oleandrin alone withrespect to the inhibition of the growth of Panc-1 or BRO cells. Incomparison, hot water extract is the least potent among three entitiestested. The data demonstrate potent cytotoxicity against human tumorcell lines by oleandrin as well as the extracts with the relativepotency occurring as follows: supercritical CO₂ extract>oleandrin>hotwater extract. These data imply that the cytotoxicity of thesupercritical CO₂ extract is probably due to the presence of at leastone other pharmacologically active component in the SCF extract inaddition to oleandrin and that the potency of the supercritical CO₂extract is much greater (7.4 fold) than that of the hot water extract.The data clearly demonstrate the substantial improvement in efficacy ofthe SCF extract over the hot-water extract and even oleandrin alone. Theimprovement in efficacy exceeded the expected improvement that mighthave been obtained based solely upon the increased concentration ofoleandrin in the SCF extract.

The invention also provides a method of inhibiting the proliferation ofcancer or tumor cells by treatment of the cells with an effective ofamount extract, such as SCF or water extract, of the invention.

The cardiac glycoside can be formulated in any suitable pharmaceuticallyacceptable dosage form. Parenteral, otic, ophthalmic, nasal, inhalable,buccal, sublingual, enteral, topical, oral, peroral, and injectabledosage forms are particularly useful. Particular dosage forms include asolid or liquid dosage forms. Exemplary suitable dosage forms includetablet, capsule, pill, caplet, troche, sache, and other such dosageforms known to the artisan of ordinary skill in the pharmaceuticalsciences.

The amount of oleandrin incorporated in a unit dose of the inventionwill be at least one or more dosage forms and can be selected accordingto known principles of pharmacy. An effective amount or therapeuticallyrelevant amount of therapeutic compound is specifically contemplated. Bythe term “effective amount”, it is understood that, with respect to, forexample, pharmaceuticals, a pharmaceutically effective amount iscontemplated. A pharmaceutically effective amount is the amount orquantity of active ingredient which is enough for the required ordesired therapeutic response, or in other words, the amount, which issufficient to elicit an appreciable biological response when,administered to a patient. The appreciable biological response may occuras a result of administration of single or multiple unit doses of anactive substance. A unit dose may comprise one or more dosage forms. Itwill be understood that the specific dose level for any patient willdepend upon a variety of factors including the indication being treated,severity of the indication, patient health, age, sex, weight, diet,pharmacological response, the specific dosage form employed, and othersuch factors.

The desired dose for oral administration is up to 5 dosage formsalthough as few as one and as many as ten dosage forms may beadministered. Exemplary dosage forms contain 0.6 mg of the SCF extractper dosage form, for a total 0.6 to 60 mg (1 to 10 dose levels) perdose.

The cardiac glycoside can be present in a dosage form in an amountsufficient to provide a subject with an initial dose of oleandrin of 12to 1200 ug, or more or less.

For use in treatment of mammals, the cardiac glycoside can be includedin a dosage form. Some embodiments of the dosage form are not entericcoated and release their charge of cardiac glycoside within a period of0.5 to 1 hours or less. Some embodiments of the dosage form are entericcoated and release their charge of cardiac downstream of the stomach,such as from the jejunum, ileum, small intestine, and/or large intestine(colon). Enterically coated dosage forms will release cardiac glycosidesinto the systemic circulation within 1-10 hr after oral administration.

Based on preliminary animal dosing data it is anticipated that 50 to 75%of an administered dose of oleander extract will be orally bioavailabletherefore providing 0.25 to 0.4 mg, 0.1 to 50 mg, 0.1 to 40 mg, 0.2 to40 mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 10 mg, 0.2 to 5 mg, 0.2 to 2.5mg, 0.2 to 2 mg, 0.2 to 1.5 mg, 0.2 to 1 mg, 0.2 to 0.8 mg, 0.2 to 0.7,or 0.25 to 0.5 mg of oleandrin per dosage form. Given an average bloodvolume in adult humans of 5 liters, the anticipated oleandrin plasmaconcentration will be in the range of 0.05 to 2 ng/ml, 0.005 to 10ng/mL, 0.005 to 8 ng/mL, 0.01 to 7 ng/mL, 0.02 to 7 ng/mL, 0.03 to 6ng/mL, 0.04 to 5 ng/mL, or 0.05 to 2.5 ng/mL. The recommended daily doseof oleandrin, present in the SCF extract, is generally about 0.25 toabout 50 mg twice daily or about 0.9 to 5 mg twice daily or about every12 hours. The dose can be about 0.5 to about 100 mg/day, about 1 toabout 80 mg/day, about 1.5 to about 60 mg/day, about 1.8 to about 60mg/day, about 1.8 to about 40 mg/day. The maximum tolerated dose can beabout 100 mg/day, about 80 mg/day, about 60 mg/day, about 40 mg/day,about 38.4 mg/day or about 30 mg/day of oleander extract containingoleandrin and the minimum effective dose can be about 0.5 mg/day, about1 mg/day, about 1.5 mg/day, about 1.8 mg/day, about 2 mg/day, or about 5mg/day.

A kit or composition of the invention can include any excipientssuitable for analytical or pharmaceutical use.

It should be noted that a compound herein might possess one or morefunctions in the formulation of the invention. For example, a compoundmight serve as both a surfactant and a water miscible solvent or as botha surfactant and a water immiscible solvent.

A liquid composition can comprise one or more pharmaceutically oranalytically acceptable liquid carriers. The liquid carrier can be anaqueous, non-aqueous, polar, non-polar, and/or organic carrier. Liquidcarriers include, by way of example and without limitation, a watermiscible solvent, water immiscible solvent, water, buffer and mixturesthereof.

As used herein, the terms “water soluble solvent” or “water misciblesolvent”, which terms are used interchangeably, refer to an organicliquid which does not form a biphasic mixture with water or issufficiently soluble in water to provide an aqueous solvent mixturecontaining at least five percent of solvent without separation of liquidphases. The solvent is suitable for administration to humans or animals.Exemplary water soluble solvents include, by way of example and withoutlimitation, PEG (poly(ethylene glycol)), PEG 400 (poly(ethylene glycolhaving an approximate molecular weight of about 400), ethanol, acetone,alkanol, alcohol, ether, propylene glycol, glycerin, triacetin,poly(propylene glycol), PVP (poly(vinyl pyrrolidone)),dimethylsulfoxide, N,N-dimethylformamide, formamide,N,N-dimethylacetamide, pyridine, propanol, N-methylacetamide, butanol,soluphor (2-pyrrolidone), pharmasolve (N-methyl-2-pyrrolidone).

As used herein, the terms “water insoluble solvent” or “water immisciblesolvent”, which terms are used interchangeably, refer to an organicliquid which forms a biphasic mixture with water or provides a phaseseparation when the concentration of solvent in water exceeds fivepercent. The solvent is suitable for administration to humans oranimals. Exemplary water insoluble solvents include, by way of exampleand without limitation, medium/long chain triglycerides, oil, castoroil, corn oil, vitamin E, vitamin E derivative, oleic acid, fatty acid,olive oil, softisan 645 (Diglyceryl Caprylate/Caprate/Stearate/Hydroxystearate adipate), miglyol, captex (Captex 350: GlycerylTricaprylate/Caprate/Laurate triglyceride; Captex 355: GlycerylTricaprylate/Caprate triglyceride; Captex 355 EP/NF: GlycerylTricaprylate/Caprate medium chain triglyceride).

Suitable solvents are listed in the “International Conference onHarmonisation of Technical Requirements for Registration ofPharmaceuticals for Human Use (ICH) guidance for industry Q3CImpurities: Residual Solvents” (1997), which makes recommendations as towhat amounts of residual solvents are considered safe inpharmaceuticals. Exemplary solvents are listed as class 2 or class 3solvents. Class 3 solvents include, for example, acetic acid, acetone,anisole, 1-butanol, 2-butanol, butyl acetate, tert-butylmethyl ether,cumene, ethanol, ethyl ether, ethyl acetate, ethyl formate, formic acid,heptane, isobutyl acetate, isopropyl acetate, methyl acetate,methyl-1-butanol, methylethyl ketone, methylisobutyl ketone,2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, orpropyl acetate.

Other materials that can be used as water immiscible solvents in theinvention include: Captex 100: Propylene Glycol Dicaprate; Captex 200:Propylene Glycol Dicaprylate/Dicaprate; Captex 200 P: Propylene GlycolDicaprylate/Dicaprate; Propylene Glycol Dicaprylocaprate; Captex 300:Glyceryl Tricaprylate/Caprate; Captex 300 EP/NF: GlycerylTricaprylate/Caprate Medium Chain Triglycerides; Captex 350: GlycerylTricaprylate/Caprate/Laurate; Captex 355: Glyceryl Tricaprylate/Caprate;Captex 355 EP/NF: Glyceryl Tricaprylate/Caprate Medium ChainTriglycerides; Captex 500: Triacetin; Captex 500 P: Triacetin(Pharmaceutical Grade); Captex 800: Propylene GlycolDi(2-Ethylhexanoate); Captex 810 D: GlycerylTricaprylate/Caprate/Linoleate; Captex 1000: Glyceryl Tricaprate; CaptexCA: Medium Chain Triglycerides; Captex MCT-170: Medium ChainTriglycerides; Capmul GMO: Glyceryl Monooleate; Capmul GMO-50 EP/NF:Glyceryl Monooleate; Capmul MCM: Medium Chain Mono- & Diglycerides;Capmul MCM C8: Glyceryl Monocaprylate; Capmul MCM C10: GlycerylMonocaprate; Capmul PG-8: Propylene Glycol Monocaprylate; Capmul PG-12:Propylene Glycol Monolaurate; Caprol 10G10O: Decaglycerol Decaoleate;Caprol 3GO: Triglycerol Monooleate; Caprol ET: Polygycerol Ester ofMixed Fatty Acids; Caprol MPGO: Hexaglycerol Dioleate; Caprol PGE 860:Decaglycerol Mono-, Dioleate.

As used herein, a “surfactant” refers to a compound that comprises polaror charged hydrophilic moieties as well as non-polar hydrophobic(lipophilic) moieties; i.e., a surfactant is amphiphilic. The termsurfactant may refer to one or a mixture of compounds. A surfactant canbe a solubilizing agent, an emulsifying agent or a dispersing agent. Asurfactant can be hydrophilic or hydrophobic.

The hydrophilic surfactant can be any hydrophilic surfactant suitablefor use in pharmaceutical compositions. Such surfactants can be anionic,cationic, zwitterionic or non-ionic, although non-ionic hydrophilicsurfactants are presently preferred. As discussed above, these non-ionichydrophilic surfactants will generally have HLB values greater thanabout 10. Mixtures of hydrophilic surfactants are also within the scopeof the invention.

Similarly, the hydrophobic surfactant can be any hydrophobic surfactantsuitable for use in pharmaceutical compositions. In general, suitablehydrophobic surfactants will have an HLB value less than about 10.Mixtures of hydrophobic surfactants are also within the scope of theinvention.

Examples of additional suitable solubilizer include: alcohols andpolyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethyleneglycol, propylene glycol, butanediols and isomers thereof, glycerol,pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide,polyethylene glycol, polypropylene glycol, polyvinylalcohol,hydroxypropyl methylcellulose and other cellulose derivatives,cyclodextrins and cyclodextrin derivatives; ethers of polyethyleneglycols having an average molecular weight of about 200 to about 6000,such as tetrahydrofurfuryl alcohol PEG ether (glycofurol, availablecommercially from BASF under the trade name Tetraglycol) or methoxy PEG(Union Carbide); amides, such as 2-pyrrolidone, 2-piperidone,caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone,N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide, andpolyvinypyrrolidone; esters, such as ethyl propionate, tributylcitrate,acetyl triethylcitrate, acetyl tributyl citrate, triethylcitrate, ethyloleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycolmonoacetate, propylene glycol diacetate, caprolactone and isomersthereof, valerolactone and isomers thereof, butyrolactone and isomersthereof; and other solubilizers known in the art, such as dimethylacetamide, dimethyl isosorbide (Arlasolve DMI (ICI)),N-methylpyrrolidones (Pharmasolve (ISP)), monooctanoin, diethyleneglycol nonoethyl ether (available from Gattefosse under the trade nameTranscutol), and water. Mixtures of solubilizers are also within thescope of the invention.

Except as indicated, compounds mentioned herein are readily availablefrom standard commercial sources.

The clear liquid composition is visually clear to the unaided eye, as itwill contain less than 5%, less than 3% or less than 1% by wt. ofsuspended solids based upon the total weight of the composition.

Although not necessary, a composition or kit of the present inventionmay include a chelating agent, preservative, antioxidant, adsorbents,acidifying agent, alkalizing agent, antifoaming agent, buffering agent,colorant, electrolyte, salt, stabilizer, tonicity modifier, diluent,other pharmaceutical excipient, or a combination thereof.

As used herein, the term “antioxidant” is intended to mean an agent thatinhibits oxidation and is thus used to prevent the deterioration ofpreparations by the oxidative process. Such compounds include, by way ofexample and without limitation, ascorbic acid, ascorbic palmitate,Vitamin E, Vitamin E derivative, butylated hydroxyanisole, butylatedhydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate,sodium metalbisulfite and other such materials known to those ofordinary skill in the art.

As used herein, the term chelating agent is intended to mean a compoundthat chelates metal ions in solution. Exemplary chelating agents includeEDTA (tetrasodium ethylenediaminetetraacetate), DTPA (pentasodiumdiethylenetriaminepentaacetate), HEDTA (trisodium salt ofN-(hydroxyethyl)-ethylenediaminetriacetic acid), NTA (trisodiumnitrilotriacetate), disodium ethanoldiglycine (Na₂EDG), sodiumdiethanolglycine (DEGNa), citric acid, and other compounds known tothose of ordinary skill in the art.

As used herein, the term “adsorbent” is intended to mean an agentcapable of holding other molecules onto its surface by physical orchemical (chemisorption) means. Such compounds include, by way ofexample and without limitation, powdered and activated charcoal andother materials known to one of ordinary skill in the art.

As used herein, the term “alkalizing agent” is intended to mean acompound used to provide an alkaline medium. Such compounds include, byway of example and without limitation, ammonia solution, ammoniumcarbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodiumborate, sodium carbonate, sodium bicarbonate, sodium hydroxide,triethanolamine, and trolamine and others known to those of ordinaryskill in the art.

As used herein, the term “acidifying agent” is intended to mean acompound used to provide an acidic medium. Such compounds include, byway of example and without limitation, acetic acid, amino acid, citricacid, fumaric acid and other alpha hydroxy acids, hydrochloric acid,ascorbic acid, and nitric acid and others known to those of ordinaryskill in the art.

As used herein, the term “antifoaming agent” is intended to mean acompound or compounds that prevents or reduces the amount of foamingthat forms on the surface of the fill composition. Suitable antifoamingagents include by way of example and without limitation, dimethicone,SIMETHICONE, octoxynol and others known to those of ordinary skill inthe art.

As used herein, the term “buffering agent” is intended to mean acompound used to resist a change in pH upon dilution or addition of acidor alkali. Such compounds include, by way of example and withoutlimitation, potassium metaphosphate, potassium phosphate, monobasicsodium acetate and sodium citrate anhydrous and dehydrate and other suchmaterials known to those of ordinary skill in the art.

As used herein, the term “diluent” or “filler” is intended to mean inertsubstances used as fillers to create the desired bulk, flow properties,and compression characteristics in the preparation of tablets andcapsules. Such compounds include, by way of example and withoutlimitation, dibasic calcium phosphate, kaolin, lactose, sucrose,mannitol, microcrystalline cellulose, powdered cellulose, precipitatedcalcium carbonate, sorbitol, and starch and other materials known to oneof ordinary skill in the art.

As used herein, the term “preservative” is intended to mean a compoundused to prevent the growth of microorganisms. Such compounds include, byway of example and without limitation, benzalkonium chloride,benzethonium chloride, benzoic acid, benzyl alcohol, cetylpyridiniumchloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuricnitrate, phenylmercuric acetate, thimerosal, metacresol, myristylgammapicolinium chloride, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, sorbic acid, thymol, and methyl, ethyl,propyl, or butyl parabens and others known to those of ordinary skill inthe art.

As used herein, the term “colorant” is intended to mean a compound usedto impart color to pharmaceutical preparations. Such compounds include,by way of example and without limitation, FD&C Red No. 3, FD&C Red No.20, FD&C Yellow No. 6, FD&C Blue No. 2, FD&C Green No. 5, FD&C OrangeNo. 5, FD&C Red No. 8, caramel, and iron oxide (black, red, yellow),other FD&C dyes and natural coloring agents such as grape skin extract,beet red powder, beta-carotene, annato, carmine, turmeric, paprika,combinations thereof and other such materials known to those of ordinaryskill in the art.

As used herein, the term “stabilizer” is intended to mean a compoundused to stabilize an active agent against physical, chemical, orbiochemical processes that would otherwise reduce the therapeuticactivity of the agent. Suitable stabilizers include, by way of exampleand without limitation, albumin, sialic acid, creatinine, glycine andother amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide,sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethyleneglycols, sodium caprylate and sodium saccharin and others known to thoseof ordinary skill in the art.

As used herein, the term “tonicity modifier” is intended to mean acompound or compounds that can be used to adjust the tonicity of theliquid formulation. Suitable tonicity modifiers include glycerin,lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol,trehalose and others known to those or ordinary skill in the art.

Examples 3 and 6 describe an exemplary capsule dosage form. Example 12describes an exemplary tablet dosage form.

The composition of the invention can also include oils such as fixedoils, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil;fatty acids such as oleic acid, stearic acid and isostearic acid; andfatty acid esters such as ethyl oleate, isopropyl myristate, fatty acidglycerides and acetylated fatty acid glycerides. The composition canalso include alcohol such as ethanol, isopropanol, hexadecyl alcohol,glycerol and propylene glycol; glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such as poly(ethyleneglycol) 450; petroleum hydrocarbons such as mineral oil and petrolatum;water; a pharmaceutically suitable surfactant, suspending agent oremulsifying agent; or mixtures thereof.

It should be understood that the compounds used in the art ofpharmaceutical formulation generally serve a variety of functions orpurposes. Thus, if a compound named herein is mentioned only once or isused to define more than one term herein, its purpose or function shouldnot be construed as being limited solely to that named purpose(s) orfunction(s).

One or more of the components of the formulation can be present in itsfree base or pharmaceutically or analytically acceptable salt form. Asused herein, “pharmaceutically or analytically acceptable salt” refersto a compound that has been modified by reacting it with an acid asneeded to form an ionically bound pair. Examples of acceptable saltsinclude conventional non-toxic salts formed, for example, from non-toxicinorganic or organic acids. Suitable non-toxic salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfonic, sulfamic, phosphoric, nitric and others known tothose of ordinary skill in the art. The salts prepared from organicacids such as amino acids, acetic, propionic, succinic, glycolic,stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and others known to those of ordinaryskill in the art. Lists of other suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th). ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, the relevant disclosure of which is herebyincorporated by reference.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith tissues of human beings and animals and without excessive toxicity,irritation, allergic response, or any other problem or complication,commensurate with a reasonable benefit/risk ratio.

In view of the above description and the examples below, one of ordinaryskill in the art will be able to practice the invention as claimedwithout undue experimentation. The foregoing will be better understoodwith reference to the following examples that detail certain proceduresfor the preparation of embodiments of the present invention. Allreferences made to these examples are for the purposes of illustration.The following examples should not be considered exhaustive, but merelyillustrative of only a few of the many embodiments contemplated by thepresent invention.

Oleandrin and MTT are purchased from Sigma Chemical Co. (St. Louis,Mo.). BODIPY-oleandrin, Mito-Tracker Red CM-H₂XRos, calceinacetoxymethyl (CAM) ester and 4′-6-diamidino-2-phenylindole (DAPI) areobtained from Molecular Probes-Invitrogen Corporation (Carlsbad,Calif.). Anti-β-actin antibody is also purchased from Sigma.

Human pancreatic cancer cells: Panc-1, BxPC3, MiaPaca; human coloncancer cell lines: CaCO-2, DOD-1, HCT-116, HT29, RKO and LST174; rodentmelanoma B16 cells; human breast cancer cells: SUM149, MCF-7 and MDA231;human oral cancer cells: SCC9 and CAL-27; human ovarian cancer ES3,TOV1120 and SKOV cells and human non-small cell lung cancer A549 andH1299 cells are obtained from the American Type Culture Collection(Manassas, Va.) and maintained in a humidified atmosphere containing 5%CO₂ at 37° C. Human melanoma BRO cells were a kind gift from the StehlinFoundation (Houston, Tex.). Cell lines derived from different epithelialorigins are routinely cultured in tissue culture medium (InvitrogenCorp., Grand Island, N.Y.) (Table 1) supplemented with 10% heatinactivated fetal bovine serum (FBS) Hyclone Laboratories Inc., Logan,Utah), 50 IU/ml penicillin and 50 μg/ml streptomycin, and 2 mML-glutamine from GIBCO (Invitrogen).

Example 1 Supercritical Fluid Extraction of Powdered Oleander Leaves

Method A. with Carbon Dioxide.

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.94 kg.

The starting material was combined with pure CO₂ at a pressure of 300bar (30 MPa, 4351 psi) and a temperature of 50° C. (122° F.) in anextractor device. A total of 197 kg of CO₂ was used, to give a solventto raw material ratio of 50:1. The mixture of CO₂ and raw material wasthen passed through a separator device, which changed the pressure andtemperature of the mixture and separated the extract from the carbondioxide.

The extract (65 g) was obtained as a brownish, sticky, viscous materialhaving a nice fragrance. The color was likely caused by chlorophyll. Foran exact yield determination, the tubes and separator were rinsed outwith acetone and the acetone was evaporated to give an addition 9 g ofextract. The total extract amount was 74 g. Based on the weight of thestarting material, the yield of the extract was 1.88%. The content ofoleandrin in the extract was calculated using high pressure liquidchromatography and mass spectrometry to be 560.1 mg, or a yield of0.76%.

Method B. with Mixture of Carbon Dioxide and Ethanol

Powdered oleander leaves were prepared by harvesting, washing, anddrying oleander leaf material, then passing the oleander leaf materialthrough a comminuting and dehydrating apparatus such as those describedin U.S. Pat. Nos. 5,236,132, 5,598,979, 6,517,015, and 6,715,705. Theweight of the starting material used was 3.85 kg.

The starting material was combined with pure CO₂ and 5% ethanol as amodifier at a pressure of 280 bar (28 MPa, 4061 psi) and a temperatureof 50° C. (122° F.) in an extractor device. A total of 160 kg of CO₂ and8 kg ethanol was used, to give a solvent to raw material ratio of 43.6to 1. The mixture of CO₂, ethanol, and raw material was then passedthrough a separator device, which changed the pressure and temperatureof the mixture and separated the extract from the carbon dioxide.

The extract (207 g) was obtained after the removal of ethanol as a darkgreen, sticky, viscous mass obviously containing some chlorophyll. Basedon the weight of the starting material, the yield of the extract was5.38%. The content of oleandrin in the extract was calculated using highpressure liquid chromatography and mass spectrometry to be 1.89 g, or ayield of 2.1%.

Example 2 Hot-Water Extraction of Powdered Oleander Leaves

Hot water extraction is typically used to extract oleandrin and otheractive components from oleander leaves. Examples of hot water extractionprocesses can be found in U.S. Pat. No. 5,135,745 and No. 5,869,060.

A hot water extraction was carried out using 5 g of powdered oleanderleaves. Ten volumes of boiling water (by weight of the oleander startingmaterial) were added to the powdered oleander leaves and the mixture wasstirred constantly for 6 hours. The mixture was then filtered and theleaf residue was collected and extracted again under the sameconditions. The filtrates were combined and lyophilized. The appearanceof the extract was brown. The dried extract material weighed about 1.44g. 34.21 mg of the extract material was dissolved in water and subjectedto oleandrin content analysis using high pressure liquid chromatographyand mass spectrometry. The amount of oleandrin was determined to be 3.68mg. The oleandrin yield, based on the amount of extract, was calculatedto be 0.26%. The table below shows a comparison between the oleandrinyields for the two supercritical carbon dioxide extractions of Example 1and the hot water extraction.

Comparison of Yields Oleandrin yield based Extraction Medium on totalextract weight Supercritical Carbon Dioxide: Example 1, 0.76% Method ASupercritical Carbon Dioxide: Example 1, 2.1% Method B Hot WaterExtraction: Example 2 0.26%

Example 3 Cell Lines

Human pancreatic cancer cells: Panc-1, BxPC3, MiaPaca; human coloncancer cell lines: CaCO-2, DOD-1, HCT-116, HT29, RKO and LST174; rodentmelanoma B16 cells; human breast cancer cells: SUM149, MCF-7 and MDA231;human oral cancer cells: SCC9 and CAL-27; human ovarian cancer ES3,TOV1120 and SKOV cells and human non-small cell lung cancer A549 andH1299 cells were obtained from the American Type Culture Collection(Manassas, Va.) and maintained in a humidified atmosphere containing 5%CO₂ at 37° C. Human melanoma BRO cells were a kind gift from the StehlinFoundation (Houston, Tex.). Cell lines derived from different epithelialorigins were routinely cultured in tissue culture medium (InvitrogenCorp., Grand Island, N.Y.) (Table 1) supplemented with 10% heatinactivated fetal bovine serum (FBS) Hyclone Laboratories Inc., Logan,Utah), 50 IU/ml penicillin and 50 μg/ml streptomycin, and 2 mML-glutamine from GIBCO (Invitrogen).

Example 4 In Vitro Determination of Cytotoxicity

Cells were grown in their relevant media as indicated in the table belowat a density of 1×10⁴ cells/well. After a 24 hr incubation period, cellswere treated with various concentrations of oleandrin (1 to 500 nM).After an additional 72 hr, inhibition of cellular proliferation wasassessed by MTT assay (23). Absorbance was read at a wavelength of 570nm and a reference wavelength of 650 nm using a V-Max Micro-plate Readerby Molecular Devices, Inc. (Sunnyvale, Calif.). The relative extent ofinhibition of cell proliferation due to the presence of a givenconcentration of oleander extract or other plant extract containing acardiac glycoside compound(s) can be derived by comparing treated tonon-treated cell numbers after a set period of time allowing for cellgrowth (e.g. 24-72 hr).

Name of cells Phenotype description Cell culture medium PANC-1 Humanpancreatic DMEM/10% FBS carcinoma BXPC3 Human pancreatic RPMI 164/10%FBS/sodium adenocarcinoma pyruvate (NaP) MiaPaca Human pancreaticDMEM/10% FBS/ adenocarcinoma 2% Equine serum MDA 231 Breast cancerDMEM/10% FBS SUM 149 Breast cancer F12/5% FBS/HEPES/insulin/hydrocortisone CaCO2 Colon carcinoma RPMI 1640/10% FBSDOD-1 Colon carcinoma RPMI 1640/10% FBS HCT 116 Colon carcinoma RPMI1640/10% FBS HT 29 Colon carcinoma RPMI 1640/10% FBS LIS-174t Coloncarcinoma MEM/10% FBS/NaP/NEAA BRO Acute lymphoblastic MEM/10% FBS/NaPleukemia SCC-9 Mouth squamous cell DMEM/10% FBS carcinoma CAL27 Mouthsquamous cell DMEM/10% FBS carcinoma MCF-7 Breast cancer MEM/10% FBS/insulin/hydrocortisone/EGF Note: DMEM = Dulbecco's Modified Eagle'sMedium; MEM = Minimum Essential Medium; FBS = Fetal bovine serum; NEAA =non-essential amino acids; EGF = Epidermal growth factor.

Example 5 Determination of Cellular Uptake of Cardiac Glycoside

Uptake of oleandrin and ouabain in Panc-1 (highest ratio of α3:α1isoforms) cells and BxPC3 cells (lower ratio of α3:α1) was determinedafter treatment with BODYPI-oleandrin, a fluorescent analog ofoleandrin, by fluorescence microscopy. Cells in 96-well plates weretreated with 0, 5, 20, and 50 nM oleandrin for 2 h or 24 h. Treatmentswere performed in 0.5% fetal bovine calf serum in DMEM/F12 medium. Cellswere simultaneously incubated with MitoTracker Red CM-H₂XRos (1 μM), andDAPI (1 ng/ml), a selective nuclear dye (Molecular Probes). Nuclearmorphology, DNA and mitochondria dye uptake were assessed byfluorescence microscopy using an Olympus IX-70 inverted microscope.Image acquisition was achieved using a Quantix charged coupled devicecamera and IP Labs software (Scanalytics, Inc., Fairfax, Va.).Alteration of oleandrin uptake in wild type and Panc-1 cells transfectedwith α3 siRNA was determined in cells cultured on laminin coatedcoverslips and treated with BODIBY-oleandrin for 1 hr.

Example 6 Determination of Na, K-ATP as α3 and α1 Isoform Expression

Cells were washed with cold PBS and scraped free in the presence oflysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 40 mMβ-glycerophosphate, 20 mM sodium pyruvate, 0.5% Triton X-100, and 1 mMsodium orthovanadate with protease inhibitor cocktail). Cell lysateswere then sonicated on ice for 3 min, incubated for an additional 10 minat 4° C. prior to centrifugation at 14,000×g (10 min at 4° C.). Proteinlevels were quantified via the BioRad Dc protein assay (BioRad, Inc.,Hercules, Calif.). Equal levels of protein (50 μg) were applied toprecast gels (BioRad) and then transferred onto polyvinylidenedifluoride membranes, according to standard methods. Following a 1- to2-hr incubation period in 5% nonfat dry milk blocking buffer prepared inTris-buffered saline with 0.1% Tween 20, membranes were probed withprimary antibodies to α3 (Affinity Bioreagents, Golden, Colo.) and α1(Upstate, Lake Placid, N.Y.) isoforms diluted 1:2,000 in blockingbuffer. Protein bands were visualized via chemluminesence using the ECL+detection kit and hyper-film (Amersham Biosciences, Piscataway, N.J.).Equal loading of samples was illustrated by Western blotting for thepresence of β-actin. Protein bands were quantified using Alpha DigiDoc1000 software (Alpha Innotech Corp., San Leandro, Calif.).

Example 7 Treatment of Skin Related Diseases Such as Cancers Includingbut not Limited to Prevention of Treatment of Melanoma, Basal CellCarcinoma, and Squamous Cell Carcinoma as Well as NoncancerousInflammatory Skin Diseases Including but not Limited to ActinicKeratosis, Psoriasis, and Eczema

The SCF extract is administered to a subject suffering from malignant ornonmalignant proliferative skin diseases such as those cited above. TheSCF extract is administered as a cream or ointment or contained within adermal patch containing 0.01 mg to 10 mg of SCF extract per unit dose.The subject is administered a unit dose up to three times daily for aperiod of 1 to 14 days or until the skin diseases is in remission. It isexpected that such treatment will significantly lessen or eliminate theinflammation and malignant processes leading to a progression of thedisease. The subject should experience a reduction in the severity ofthe dermal lesion(s) and the eventual resolution of the dermatologicdisease itself. Malignant diseases should be expected to be reduced inrate of growth or inhibited from increase in severity of the disease.Actual regression of established malignant lesions may be expected.

Example 8 Prevention of Skin Related Diseases Such as Skin Cancers

The SCF extract is administered to a subject suffering from apredisposition to formation of skin cancer such as those frequentlyexposed to ultraviolet light (from sunlight) or carcinogens fromchemicals. The SCF extract is administered as a cream or ointment orcontained within a dermal patch containing 0.01 to 10 mg of SCF extractper unit dose. The subject is administered a unit dose up to three timesdaily every time exposure to a carcinogen promoting event is anticipated(exposure to sunlight). Such administration could, for example, be madeas a sunscreen for blocking sunlight UV exposure and SCF extract forprevention of tumor induction in dermal tissue. It would be expectedthat such a use of the SCE in a dermal product would block formationand/or promotion of malignant skin disease or nonmalignant skindisorders where proliferation leads to a worsening of the diseaseprocess (e.g. acktinic keratosis, psoriasis and/or eczema).

Example 9 Treatment of Solid Tumors in Humans or Other VertebrateAnimals

SCF extract of plants or animals containing cardiac glycosides can beused to treat cancers of the rectum, anus, colorectal tissues, head andneck tissues, esophageal tissue, lung (both non small cell and smallcell carcinomas), breast, stomach, pancreas, prostate, liver, kidney,bladder, ureter, ovarian tissue, carcinoid tumors, sarcomas of bone,mesothelioma, and neoplasms of the central nervous system.

The SCF extract is administered to a subject suffering from solidmalignant diseases such as those mentioned above. The SCF extract isadministered as an oral dosage form containing 1 to 50 mg of SCF extractper unit dose. The subject is administered a unit dose up to twice dailytimes daily for a period of 28 days/cycle of treatment. Up to threecycles of treatment may be required. The subject should experience tumorgrowth to either slow in rate of proliferation or to regress. Completionresolution of the tumor may occur. The therapy with SCF extract may beused as a sole agent or combined with cytotoxic chemotherapy orradiation treatment or may be combined with appropriate immunotherapywithout causing undue interference with the desired antitumor effect ofconventional therapy.

Example 10 Comparison of Cytotoxicity of Hot Water Extract of Neriumoleander to an SCF Extract Made Using Supercritical CO₂ in Two HumanTumor Cell Lines

The cytotoxic potential of both extracts are compared directly with thatof oleandrin. The samples contained the same amounts of oleandrin eventhough their concentration of oleandrin differed due to theconcentration of oleandrin present in the extracts.

BRO (human melanoma) and Panc-1 (human pancreatic cancer) cells(8×10³/well) were plated in a 96 well plate and allowed to attachovernight. Drug or extracts were then added to the cells. After 72 hr ofincubation, relative cell proliferation (relative to control untreatedcells) was assessed by crystal violet staining method.

Example 11 HPLC Analysis of Solutions Containing Oleandrin

Samples (oleandrin standard, SCF extract and hot-water extract) wereanalyzed on HPLC (Waters) using the following conditions: Symmetry C18column (5.0 μm, 150×4.6 mm I.D.; Waters); Mobile phase ofMeOH:water=54:46 (v/v) and flow rate at 1.0 ml/min. Detection wavelengthwas set at 217 nm. The samples were prepared by dissolving the compoundor extract in a fixed amount of HPLC solvent to achieve an approximatetarget concentration of oleandrin.

Example 12 Evaluation of Anti-Viral Activity of an SCF Extract

The test consists of determining the relative ability of oleanderextract or a positive control (AZT) to inhibit proliferation of the ROJOstrain of HIV-1 in human peripheral blood mononuclear cells (PBMCs).Infected cells are exposed to the drug or extract for 48 hr. The test isused to determine the IC₅₀ of oleander extract (that concentration ofextract producing a 50% inhibition of viral proliferation) versus thatconcentration of extract capable of killing the human PBMC. This is, ineffect, a determination of the therapeutic index of the extract. This isessentially a determination of whether or not the extract can kill HIV-1without killing the PBMC cell itself.

One should observe an IC₅₀ against viral proliferation of about 5.0ug/ml or less while the concentration required to kill cells should nothave been reached even at concentrations as high as 100 ug/ml. The dataobtained suggest that oleander extract should be useful in terms ofinhibiting HIV-1 viral proliferation or infectivity of virus harboredwithin PBMC cells.

Example 13 Transfection of Panc-1 Cells with α3 siRNA

Panc-1 cells were plated in 6 and 48 well plates and allowed to attachovernight. Transient transfection of α3 siRNA molecules was carried outusing siPORT™ Amine Transfection Agent (Ambion Austin, Tex.) and 0.4 μMα3 silencing RNA (Santa Cruz Biotech.) following the manufacture'sinstructions. Twenty-four hr after transfection, cells were treated with10 to 50 nM oleandrin for 48 hr. Protein was collected from the 6 wellplates for Western blot analysis and cell viability assessment wascarried out by Calcien AM staining.

Example 14 Determination of α3 and α1 Expression in Normal and ColonBiopsy Tissues

Flash frozen normal colon mucosa and tumor tissue biopsies were obtainedand were pulverized with use of a liquid nitrogen cooled mortar. Sampleswere exposed to lysis buffer as indicated above. Lysates were thensonicated on ice for 3 min., incubated at 4° C. for 10 min. andcentrifuged at 14,000 rpm (10 min. at 4° C.) followed by Westernblotting analysis of α3 and α1 isoforms of the α-subunit as describedpreviously.

Example 15 Statistical Analysis

Student's t test was used to determine the statistical differencesbetween various experimental groups; a value of P<0.05 was considered tobe significant.

Example 16 Determination of the Sensitivity of α3 Antibody

To compare the sensitivity of different α3 antibodies, equal amounts ofthe same lysates of positive and negative control samples were loadedonto 3 precast gels (BioRad, Inc., Hercules, Calif.) and thentransferred to polyvinylidene difluoride membranes, according tostandard methods. Following a 1-2 hr incubation in 5% nonfat dry milkblocking buffer prepared in tris-buffered saline with 0.1% tween 20(TBS-T), membranes were probed with α3 antibodies from Sigma (St Louis,Mo.), from Affinity Bioreagents (Golden, Colo.) and from Novus(Littleton, Colo.), each at a 1:2000 dilution in blocking buffer.Protein bands were visualized via chemiluminescence, using the ChemiglowWest detection kit and Alpha Imager (Alpha InnotechCorp., San Leandro,Calif.). Protein bands were quantified and compared between the threeantibodies, using Alpha DigiDoc 1000 software (Alpha Innotech Corp., SanLeandro, Calif.). Equal loading of samples was illustrated by Westernblotting for β-Actin. The data indicate that sensitivity of each one ofthe selected antibodies to α3 is substantially the same.

Source of α3 monoclonal antibodies Average density* Affinity Bioreagents45174 Novus 41110 Sigma 44572 *Average density was calculated based onthe absolute density of each band divided by the defined area which isquantified by Alpha DigiDoc 1000.

Example 17 Kit for Conducting Prognostic Assay

This kit can be used according to the method of the invention to conducta prognostic assay using a Western blot technique, such as detailed inExample 18.

1. Lysis composition

From Stock solution 5 ml concentration of: 20 mM Tris HCL pH 8.0 100 ul 1M 137 mM NaCl 685 ul  1M 10% Glycerol 500 ul  5 mM EDTA 25 ul 1M 1%NP-40 50 ul Protease Inhibitor cocktail 50 ul (Sigma, Cat# P8340) 1 mMNa Orthovanadate 50 ul 100 mM H₂O 3540 ul 

2. Primary antibodies: (1:2000 in blocking solution)

-   -   Alpha 3 (Affinity Bioreagents Cat #MA3-915)    -   Alpha 1 (Upstate Cat #05-369)

3. Secondary antibody: Goat anti-mouse IgG HRP (Santa Cruz Cat #sc-2005)

4. Gel/Membrane Prep:

-   -   7.5-10% Gels (BioRad Precast)    -   BioRad Laemmli sample buffer (Cat #161-0737)    -   Molecular weight marker (Cat #161-0318)    -   Running buffer (BioRad 10× Tris/glycine/SDS) (Cat #161-0732)    -   Membranes (Biorad PVDF)    -   Transfer buffer:

3.03 g Tris (final concentration - 25 mM) 14.4 g Glycine (finalconcentration - 0.192 mM)  200 ml Methanol (20%) up to 1 L H₂O

5. Blocking solution: 5% milk in TBS (20 mM Tris HCl, 150 mM NaCl)

6. Wash buffer: TBS-T (20 mM Tris HCl, 150 mM NaCl, 0.05% Tween-20)

8. Positive control cells: cell lysate of human pancreatic cancer Panc-1cells

9. Negative control cells: Mouse melanoma B16 cells

10. Brochure including the detailed protocol for western blottinganalysis of the isoforms of the α-subunit.

Example 18 Preparing Samples of Cellular Tissue

Method A. from Cell Pellet

Obtain a cell pellet containing 2 to 4 million cells. Rinse withphysiologic balanced solution (PBS) to remove media. Add 100 ul lysisbuffer. Keep cold (over ice) at all times. Continue to sonication stepin Example 18.

Method B. from Cells on Plate

Grow 1.2 to 1.5 million cells on a 100 mm tissue culture plates for 24hrs. Carefully decant and discard cell growth media. Rinse plate 2 timeswith 1 ml PBS. Add 100 ul lysis buffer, scrape cells from plate, andcollect in tubes. Keep cold (over ice) at all times. Continue tosonication step in Example 18.

Method C. from Tissue

Grind frozen tissue. Place ground tissue in tubes. Add 100 μl of coldlysis buffer to a minimum of 5 mg of ground tissue. Keep cold (over ice)at all times. Continue to sonication step in Example 18.

Example 19 Determining Content of Isoforms of the α-Subunit in Sample byWestern Blot

The following procedure is merely one way in which the method of theinvention can be employed to detect and quantify the content of theisoforms of the α subunit of Na, K-ATPase in a sample. The order of thesteps can be modified as needed.

Western Blot Analysis

-   -   1. Sonicate cell lysate while cooling, such as with an        ice-chilled bath.    -   2. Spin lysate @ 4° C. for 10 min at 14,000 rpm.    -   3. Collect the supernatant—keep on ice at all times.    -   4. Determine the protein levels by protein assay. (See BioRad®        Protein Assay Kit directions)    -   5. Prepare samples for loading on gel based on protein assay (50        μg of protein per well).        -   a. Prepare a positive and negative control lysate along with            the samples.            -   i. Positive control: Panc-1 protein lysate            -   ii. Negative control: Panc-2 or B16 protein lysate        -   b. Add laemmli sample buffer (LSB) to samples and controls            (see LSB directions)        -   c. Heat samples and controls @ 95 C for 5 min.        -   d. Spin down samples    -   6. Load gels.    -   7. Run gel at 200 volts until the dye runs off the bottom of the        gel.    -   8. Transfer at 100 volts for 1-2 hours.    -   9. Block membrane 30 min to 1 hr in blocking buffer@ RT.    -   10. Incubate membrane overnight at 4° C.    -   11. Wash membrane with wash buffer:        -   3 quick washes, 1-15 min wash, 2-10 min washes    -   12. Incubate membrane for 45 min to 1 hr in II° antibody at RT.    -   13. Wash membrane with wash buffer:        -   3 quick washes, 1-15 min wash, 2-10 min washes    -   14. Incubate membrane for 5 min in ECL+ (Amersham Cat #RPN2132).    -   15. Expose membrane to film (Amersham Cat #RPN3114k) and develop        on film developer machine.    -   16. Take a picture of the exposed film with the Alpha Imager,        using Alpha Digi Doc software (or your current imager).    -   17. Using the Spot Denso application of the Analysis tools in        Alpha Digi Doc, select the corresponding bands of the Alpha 1        and Alpha 3 images.    -   18. Obtain the Integrated Density Value (IDV) for each band.    -   19. Calculate the ratio of α3 isoform to α1 isoform.

Example 20 RT-PCR Method for Determination of mRNA for Na,K-ATPaseSpecific Alpha Subunits

The RNA STAT-60 reagent (Tel-Test, Inc., Friendswood, Tex.) was used toextract the total RNA, which was treated with DNase I prior to use in areverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Onemicrogram of RNA was reverse transcribed with mouse mammary tumor virusRT (Life Technologies, Inc., Rockville, Md.). α3-371 bp sequences wereamplified by primer set α3-NKA3VSAS-2-F 5′-NNNNNNNNNN-3′ (forward) (SEQID NO. 3) and -R 5′-NNNNNNNN-3′ (reverse). These sets and additionalprimer sets were designed and verified using Oligo 6.7 Molecular BiologyInsights (Cascade, Colo.). Primer pairs (5′-CAGCTCTGGAGAACTGCTG-3′ (SEQID NO. 1); 5′-GTGTACTCAGTCTCCACAGA-3′ (SEQ ID NO. 2)) were used inRT-PCR analysis to detect GAPDH mRNA.

Example 21 Clinical Evaluation of the Combination of a CardiacGlycoside-Containing Extract with Other Anticancer Drugs for theTreatment of Metastatic Pancreatic Gastrinoma

The following is a case history wherein a patient suffering frommetastatic pancreatic gastrinoma was treated with a cardiacglycoside-containing extract.

A sixty three year old male presented to clinic at M.D.Anderson CancerCenter on Aug. 30, 2002 with suspected pancreatic disease. CT Scanrevealed mass on tail of pancreas. On Sep. 17, 2002 patient began toself-administer an experimental drug containing cardiac glycosides,principally oleandrin. On Oct. 9, 2002 patient was diagnosed withwell-differentiated islet cell tumor of the pancreas. Patient wasrecommended for eight cycles of chemotherapy of Adriamycin,streptozocin, and 5-FU followed by three cycles of streptozocin and5-FU. On Nov. 13, 2002, patient began recommended therapy whilecontinuing to self-administer the drug containing oleandrin. On Jul. 23,2003, patient concluded the recommended regimen of chemotherapy. Nochange was noted in his original diagnosis. Patient received nochemotherapy subsequent to Jul. 23, 2003. Patient continued toself-administer the extract containing cardiac glycosides subsequent tothe conclusion of the chemotherapy regimen. Patient is described as lateas Feb. 16, 2007 as having radiologically stable disease. Patientcontinues to self-administer the extract containing cardiac glycoside,such as oleandrin, and is asymptomatic and reports 100% on a Karnofskyscale.

Example 22 Clinical Evaluation of a Cardiac Glycoside-Containing Extractfor the Treatment of Adenocarcinoma

A 35-year-old man experienced pain and bloating in his abdomen followingeating regular meals. He presented to a private clinic, and spiralabdomen computer aided tomography (CAT) scan was performed. Among theconclusions was volumetric increase of the pancreas head, heterogeneityin the density of parenchyma, and multiple lymphadenopathies at theanterior site of the pancreas head. Magnetic resonance imaging (MRI)examination on confirmed these findings. Ultrasound aided thin needleaspiration biopsy was performed on the same day. Ultrasound examinationdemonstrated a tumoral mass of 39×33 mm in the pancreas head.Histopathological examination of the biopsy specimen revealed thediagnosis as adenocarcinoma. A course of chemotherapy was recommended,but the patient declined.

A month later, the patient began a self-administered botanical extracttherapy that contained very small quantities of oleandrin. Upper abdomenMR images obtained within a month demonstrated a contrast retaininglesion of 35×25 mm on the head of the pancreas. Numerouslymphadenopathies, of which the largest one was 5 mm, were also revealedat the anterior site of the pancreas head and the stomach antrumposterior site.

MRI images obtained three months later showed that the tumor in thepancreas head measured 30×25 mm. Dosage of the extract containing smallamounts of oleandrin was increased at this time. A bone scintigraphy wasperformed three weeks later. It was unremarkable for metastatic disease.Upper and lower abdominal MRI images obtained seven weeks laterdemonstrated that the tumoral mass in the head of pancreas and all thelymphadenopathies were indicative of remission. An upper and lowerabdominal MRI obtained two months later, was unremarkable for pancreaticadenocarcinoma and/or lymphadenopathies. A follow up upper and lowerabdominal MRI was performed four months later. It continued to beunremarkable for pancreatic adenocarcinoma and metastatic disease.

As of March 2007, the patient continues in remission.

Example 23 Culture of Cancer and Tumor Cells

The following procedure can be modified as needed to optimize theculture of specific cancer or tumor cells. Panc-1 human pancreaticcancer cells were purchased from the American Type Culture Collection(Manassas, Va.). Cells were cultured in DMEM media supplemented with10-15% fetal bovine serum (Invitrogen, Carlsbad, Calif.), 100 U/ml ofpenicillin (Invitrogen), and 2.5 ug/ml of antimycotic (Fungizone;Invitrogen) at 37° C. in 5% CO₂. Relative inhibition of cellproliferation by oleandrin and Adriamycin was determined after 72 hr ofcontinuous drug exposure of a series of concentrations of each drug. TheMTT assay was used as previously described (Mosmann, 1983) to assesscell growth relative to untreated Panc-1 cell proliferation. Cells wereexposed to no treatment (controls), oleandrin or Adriamycin for 72 priorto assessment of cell growth.

Example 24 Cell Staining

The following procedure was used to stain cells with acridine orange.

Cells were stained with 1 ug/ml acridine orange for 15 min. at 37° C.Cells were washed with PBS, trypsinized from the plates, collected inPBS with FBS, and analyzed via flow cytometry.

Example 25 Cell Cycle Analysis

The following procedure was used to determine cell cycle.

Panc-1 cells were treated with oleandrin (0, 20 and 40 uM) for 72 hr,trypsinized, fixed with 4° C. 70% ethanol, stained with propidium iodideby using a cellular DNA flow cytometric analysis reagent set (Roche),and then analyzed for DNA content by FACScan (Becton Dickinson, SanJose, Calif.). Data were analyzed by Cell Quest software (BectonDickinson). At least 100,000 cells were analyzed for each sample.

Example 26 Clinical Evaluation of a Cardiac Glycoside-Containing Extractfor the Treatment of Non-Small Cell Lung Cancer

An eighty one year-old male was diagnosed with non small cell lungcancer of the left upper lobe. Patient was recommended for radiationtherapy followed by a course of chemotherapy. Seven weeks later, thepatient began the radiation therapy and completed therapy in 24 days.The patient then commenced a regimen of five chemotherapeutic within sixdays and completed them eight months later. Twenty seven days prior tocompletion, he presented to clinic for restaging of prior diagnosis ofnon small cell lung cancer of the left upper lobe. A whole body PET scanwas performed and was conclusive for a large area of abnormal hypermetabolic activity involving the left upper lobe consistent withpatient's known malignancy. Additionally, subtle areas of hypermetabolic activity involving the right hilum and anterior mediastinumlikely reflected nodal involvement. A CT scan of the chest confirmed a5-cm soft-tissue density in the upper left lobe. A subsequentchemotherapeutic regimen was initiated. Within one month, chemotherapywas discontinued due to intolerance by patient. A week later, a CT scanof the chest was performed for comparison. There was an approximately 4cm soft-tissue mass in the posterior segment of the left upper lobe.There was an interval decrease in overall bulk of left upper lobe mass.Within five weeks, the patient began self-administration of a botanicalextract containing oleandrin. After three months, a follow-up chest PA &Lateral was compared with the prior scan, and the tumor mass density inthe left upper lobe was unchanged. A year later a chest PA & Lateral wascompared to the findings of a scan of three months prior, and it wasdetermined that the tumor mass density measuring about 4 cm in the leftupper lobe above the hilum was unchanged.

The patient continued to self-administer the botanical extractcontaining oleandrin. Within four months, a CT scan of chest with IVcontrast was conducted. An approximately 20% decrease in size of centralmass of upper left lobe was observed. At present date, the patientcontinues to self-administer the botanical extract containing oleandrinand reports high Karnofsky score. Clinical data suggests that thebotanical extract has therapeutic benefit, since the changes in the sizeof the central mass occurred many months after patient's lastchemotherapy.

Example 27 Determining Content of Isoforms of the α-Subunit in Sample byImmunohistochemical Staining

The following procedure is merely an alternative way in which the methodof the invention can be employed to detect and quantify the content ofthe isoforms of the α subunit of Na, K-ATPase in a sample. The order ofthe steps can be modified as needed. The materials used in this assaycan be obtained from Vector Laboratories (Burlingame, Calif. orPeterborough, England).

Immunohistochemical Analysis

Fresh frozen sections of human tissues were used. Sections weredeparaffinized with 3 changes of xylene (5 min each), rehydrated in adescending ethanol series (99%×2, 90%×2 for 5 min each), and rinsed wellin distilled water. Heat mediated antigen retrieval methodology wascompleted by incubation of sections for 3×5 min in a boiling solution ofVector antigen unmasking solution (Vector-AMS), high pH (VectorLaboratories Catalog #H-3301).

Following antigen retrieval, sections were washed in 50 mM Tris HCl, 300mM NaCl, 0.1%, pH 7.6 (TBS) for 2×5 min. Endogenous peroxide activitywas quenched by incubating the sections for 20 min in 0.3% 9 v/v)hydrogen peroxide in methanol, followed by washing in TBS for 10 min.

Sections were incubated for 1 hour with anti-Na, K-ATPase α3 subunitisoform antibody (Sigma-Aldrich Cat # A273) diluted in 2.5% (v/v) normalhorse serum (2.5% NHS) in TBS at 4 ug/ml. The negative control sectionswere incubated with a non-immune mouse IgG1 antibody (BiostatDiagnostics, Cat #093101) at 4 ug/ml or in 2.5% NHS (‘no primary’control).

Following washing in TBS for 2×5 min, the sections were incubated withVector ImmPress™ universal antibody reagent (a mixture of anti-rabbitIgG and anti-mouse IgG reagents; Vector Laboratories Ltd., Cat #MP-7500)for 30 min. The sections were then washed for 2×5 min and incubated withdiaminobenzidine (DAB) substrate, with monitoring until a suitable levelof staining had developed. The chromagenic reaction was stopped byimmersing the slides in distilled water.

Following chromagenesis, the sections were counterstained withhaematoxylin, dehydrated in an ascending series of ethanol solutions(90-99-100%), cleared in two changes of xylene and coverslipped underDePeX.

An assay control demonstrating cytokeratin immunoreactivity in colonmucosa was included to validate the ImmPress™ and chromagenic reagents.A ‘no primary’ control was included to assess non-specific binding ofthe secondary antibody and other assay reagents. Stained sections wereanalyzed, and suitable digital images were captured using an OlympusBX51 microscope with a Leica DFC290 camera.

Photographs of immunohistochemically stained cells are depicted in FIGS.6A-6F and 7A-7H. Quantitation of the isoforms of the subunit can beaccomplished as described herein. Following determination of the ratioof α3 isoform to α1 isoform content in the sample, a determination ismade as to the likelihood of a therapeutic response to treatment withcardiac glycoside.

The term “about” is intended to mean±10%, ±5%, ±2.5% or ±1% relative toa specified value, i.e. “about” 22% means 22±2.2%, 22±1.1%, 22±0.55% or22±0.22%.

The above is a detailed description of particular embodiments of theinvention. It will be appreciated that, although specific embodiments ofthe invention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims. All of the embodiments disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure.

1. An in vitro prognostic assay useful for predicting the in vivotherapeutic responsiveness of a disease or disorder, having an etiologyassociated with excessive cell proliferation, to treatment with acardiac glycoside or composition comprising a cardiac glycoside, theassay comprising: determining the ratio of α3 isoform to α1 isoform ofNa, K-ATPase α-subunit in a sample obtained from diseased in vivocellular tissue of a subject with a disease or disorder having anetiology associated with excessive cell proliferation, the samplecomprising one or more isoforms of the α-subunit of Na, K-ATPase,wherein the step of determining said ratio comprises quantifying thelevel of expression of each of the α3 subunit isoform of Na, K-ATPaseand the α1 subunit isoform of Na, K-ATPase in the in vitro sample orbiopsy sample, and calculating the ratio thereof; or the step ofdetermining the ratio comprises determining the amount of each of the α3subunit isoform of Na, K-ATPase relative to amount of the α1 subunitisoform of Na, K-ATPase in the in vitro sample, and calculating theratio thereof; and determining the probability of a therapeutic responseaccording to said ratio of α3 isoform to α1 isoform of Na, K-ATPase inthe subject were the subject to be treated with a therapeuticallyrelevant dose of said cardiac glycoside according to a prescribed dosingregimen, wherein the probability is determined according to thefollowing table: Probability that there will be a therapeutic Ratioresponse in the subject 0.3-0.45 +/− 0.05 20-<30% 0.5-0.95 +/− 0.0530-50%   >/=1 +/− 0.05 >50% >10  >75%.


2. The assay of claim 1 further comprising predicting that diseasedtissues having an α-subunit isoform ratio within the range of 1 to 100will be more therapeutically responsive than those having an α-subunitisoform ratio less than
 1. 3. The assay of claim 1 further comprisingpredicting that those tissues with only detectable α3 isoform and nodetectable α1 isoform will be the most therapeutically responsive tocardiac glycosides.
 4. The assay of claim 1 further comprisingpredicting that the cellular tissue will be at least partiallytherapeutically responsive to treatment with a cardiac glycoside if theratio is ≧25; or further comprising predicting that the cellular tissuewill be at least partially therapeutically responsive to treatment witha cardiac glycoside if the ratio is ≧75.
 5. The assay of claim 1 furthercomprising conducting a statistical analysis on data from which theratio is determined.
 6. The assay of claim 1, wherein the sample iscellular tissue, cellular mass, cellular lysate, membrane preparationsprepared from these, or fixed histopathology slides thereof.
 7. Theassay of claim 1, wherein the sample is an in vitro sample.
 8. The assayof claim 1, wherein the sample comprises at least two isoforms of the ccsubunit of Na, K-ATPase.
 9. The assay of claim 8, wherein the samplecomprises at least the α1 and α3 isoforms of the cc subunit of Na,K-ATPase.
 10. The assay of claim 1 further comprising: lysing ordisrupting cells, tissues or biopsy samples; or fixing tissue sectionsfor histopathologic examination from diseased in vivo cellular tissue toform the sample.
 11. The assay of claim 1 further comprising: performinga Western blot assay and/or immunohistochemical staining assay on thesample to determine the amount and relative expression of α3 subunitisoform of Na, K-ATPase relative to the α1 subunit isoform of Na,K-ATPase in the sample; and calculating the ratio thereof.
 12. The assayof claim 1 further comprising: conducting a radiometric or densitometricanalysis of a gel in order to determine the content of α3 subunitisoform of Na, K-ATPase relative to the content of α1 subunit isoform ofNa, K-ATPase in the sample.
 13. The assay of claim 1 further comprising:conducting a radiometric or densitometric analysis of a gel in order todetect the presence of and quantify the content of α3 subunit isoform ofNa, K-ATPase and of α1 subunit isoform of Na, K-ATPase in the sample.14. The assay of claim 1 further comprising: comparing the content of α3subunit isoform of Na, K-ATPase and of α1 subunit isoform of Na,K-ATPase in the sample relative to the content of α3 subunit isoform ofNa, K-ATPase and/or of α1 subunit isoform of Na, K-ATPase in a positivecontrol sample and/or a negative control sample.
 15. The assay of claim1 further comprising: comparing the content of α3 subunit isoform of Na,K-ATPase and of α1 subunit isoform of Na, K-ATPase in a tissue samplewhere expression of only one of α3 and α1 subunit is known to occur as acontrol.
 16. The assay of claim 1, wherein the diseased cellular tissueis obtained from a subject such as a mammal.
 17. The assay according toclaim 16, wherein the diseased cellular tissue is obtained from a human,cow, dog, cat, horse, pig or other domesticated animals whether ofcommercial value or not.
 18. The assay of claim 1, wherein the diseaseor disorder having an etiology associated with excessive cellproliferation is cancer or tumor or other proliferative diseases thatimpact adversely on human or animal quality of life.
 19. The assayaccording to claim 18, wherein the cancer or tumor is selected from thegroup consisting of colorectal cancer, head and neck cancer, adrenalcortical cancer, anal cancer, bile duct cancer, bladder cancer, bonecancer, bone metastasis, sarcomas of bone, brain cancer, breast cancer,cervical cancer, non-Hodgkin's lymphoma, rectal cancer, esophagealcancer, eye cancer, gallbladder cancer, gastrointestinal carcinoidtumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi'ssarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, livercancer, lung cancer (both non small cell and small cell carcinomas),lung carcinoid tumors, malignant mesothelioma, metastatic cancer,multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasalcancer, nasopharyngeal cancer, neuroblastoma, neoplasms of the centralnervous system, oral cavity and oropharyngeal cancer, osteosarcoma,ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer,prostate cancer, retinoblastoma, salivary gland cancer, sarcoma, skincancer, stomach cancer, testicular cancer, thymus cancer, thyroidcancer, cancer of the ureter; uterine sarcoma, vaginal cancer, vulvacancer and Wilm's tumor.
 20. The assay of claim 1 further comprising:identifying a subject having a disease or disorder having an etiologyassociated with excessive cell proliferation.
 21. The assay according toclaim 20 further comprising: obtaining a sample of diseased cells fromthe subject.
 22. The assay of claim 1 further comprising: includinginformation specifying how to perform analyses for the α1 and α3isoforms of the α-subunit of Na,K-ATPase.
 23. The assay of claim 1further comprising: including information detailing how to interpretprognostic data.
 24. The assay of claim 1, wherein the disease ordisorder having an etiology associated with excessive cell proliferationis selected from the group consisting of: 1) autoimmune diseases such asantigen-induced arthritis and allergic encephalomyelitis; 2) chronicinflammatory proliferative diseases such as rheumatoid arthritis,systemic-onset juvenile chronic arthritis, osteoporosis, and psoriasis;3) proliferative diseases of the breast including fibrocystic disease;4) proliferative diseases of the prostate including benign prostatichyperplasia (BPH); 5) proliferative diseases of the eye includingproliferative diabetic retinopathy; and 6) vascular proliferativediseases including atherosclerosis and coronary stenosis.
 25. The assayof claim 1, wherein the cardiac glycoside is present in pure formwhether derived through extraction of a plant or animal source,synthesized or manufactured through chemical modification of anavailable cardiac glycoside.
 26. The assay according to claim 1, whereinthe cardiac glycoside is present in an extract.
 27. The assay accordingto claim 26, wherein the cardiac glycoside extract was prepared bysupercritical fluid (SCF) extraction optionally in the presence of amodifier.
 28. The assay according to claim 27, wherein the SCF extractfurther comprises at least one other pharmacologically active agentaside from the cardiac glycoside.
 29. The assay according to claim 28,wherein the other active agent may contribute to the therapeuticefficacy of the cardiac glycoside when the extract is administered to asubject.
 30. The assay according to claim 29, wherein the other activeagent functions additively or synergistically to contribute to thetherapeutic efficacy of the cardiac glycoside.
 31. The assay accordingto claim 25, wherein the cardiac glycoside is present in apharmaceutical formulation or composition.
 32. The assay according toclaim 25, wherein the cardiac glycoside is selected from the groupconsisting of oleandrin, ouabain, bufalin, digitoxin, digoxin,cinobufatalin, cinobufagin, and resibufogenin.
 33. The assay accordingto claim 25, wherein the cardiac glycoside has been obtained from anoleander plant mass.
 34. The assay according to claim 33, wherein theoleander plant mass comprises Nerium species, such as Nerium oleander,or of Thevetia species, such as Thevetia nerifolia.
 35. The assayaccording to claim 26, wherein the extract has been obtained from toadskin or secretions derived therefrom.
 36. The invention of claim 1comprising alternative means of determining relative Na,K-ATPaseα-subunit composition and isoform ratios, wherein the alternative meanscomprises: antibodies in an enzyme linked immunoabsorbant assay orprotein tissue or cell lysate array; Northern blot analyses formeasurement of mRNA to different Na,K-ATPase subunit isoforms; orimmunohistochemical staining assay.
 37. A method of determining theprobability that a disease or disorder having an etiology associatedwith excessive cell proliferation, in a subject having such disease ordisorder, will be at least partially therapeutically responsive totreatment with a cardiac glycoside, the method comprising: a)determining the ratio of α3 isoform to α1 isoform of Na, K-ATPase in asample obtained from diseased cellular tissue of the subject, the samplecomprising one or more isoforms of the α-subunit of Na, K-ATPase,wherein the step of determining said ratio comprises quantifying thelevel of expression of each of the α3 subunit isoform of Na, K ATPaseand the α1 subunit isoform of Na, K-ATPase in the in vitro sample orbiopsy sample, and calculating the ratio thereof; or the step ofdetermining the ratio comprises determining the amount of each of the α3subunit isoform of Na, K-ATPase relative to amount of the α1 subunitisoform of Na, K-ATPase in the in vitro sample, and calculating theratio thereof; and b) determining the probability of a therapeuticresponse according to said ratio of α3 isoform to α1 isoform of Na,K-ATPase in the subject were the subject to be treated with atherapeutically relevant dose of said cardiac glycoside, wherein theprobability that there will be at least a partial therapeutic responseis related to the ratio of α3 isoform to α1 isoform of Na, K-ATPaseaccording to the following table: Probability that there will be atleast a Ratio partial therapeutic response in the subject 0.3-0.45 +/−0.05 20-<30% 0.5-0.95 +/− 0.05 30-50%   >/=1 +/− 0.05 >50% >10  >75%.


38. The method of claim 37, wherein the step of determining the ratiocomprises quantifying the level of expression of each the α3 subunitisoform of Na, K-ATPase and the α1 subunit isoform of Na, K-ATPase inthe in vitro sample or biopsy sample, and calculating the ratio thereof.39. The method of claim 38 further comprising: conducting a statisticalanalysis on data from which the ratio is determined.
 40. The method ofclaim 39 further comprising: lysing or disrupting cells, tissues orbiopsy samples; or fixing tissue sections for histopathologicexamination from diseased in vivo cellular tissue to form the sample.41. The method of claim 40 comprising: performing a Western blot assayor immunohistochemical staining assay on the sample to determine theamount and relative expression of α3 subunit isoform of Na, K-ATPase andof the α1 subunit isoform of Na, K-ATPase in the sample; and calculatingthe ratio thereof.
 42. The method of claim 41 further comprising:comparing the content of α3 subunit isoform of Na, K-ATPase and of α1subunit isoform of Na, K-ATPase in the sample relative to the content ofα3 subunit isoform of Na, K-ATPase and/or of α1 subunit isoform of Na,K-ATPase in a positive control sample and/or a negative control sample.43. The method of claim 42 further comprising: identifying a subjecthaving a disease or disorder having an etiology associated withexcessive cell proliferation.
 44. The method of claim 43 furthercomprising: obtaining a sample of diseased cells from the subject. 45.The method of claim 44 further comprising: providing informationspecifying how to perform analyses for the α1 isoform and α3 isoform ofthe α-subunit of Na,K-ATPase.
 46. The method of claim 45 furthercomprising: providing information detailing how to interpret prognosticdata.
 47. The method of claim 41, wherein the Western blot assaycomprises: a) a first primary antibody having a binding affinity for theα3 subunit isoform of Na, K-ATPase; and b) a second primary antibodyhaving a binding affinity for the α1 subunit isoform of Na, K-ATPase.48. The method of claim 42 comprising: conducting a radiometric ordensitometric analysis of a gel in order to determine the content of α3subunit isoform of Na, K-ATPase and the content of α1 subunit isoform ofNa, K-ATPase in the sample; and determining the ratio of α3 isoform toα1 isoform present in the sample.
 49. The method of claim 41, whereinthe immunohistochemical staining assay comprises: a) antigen unmaskingsolution; b) buffer; c) endogenous-peroxide activity-quenching material;d) anti-Na,K-ATPase α3 subunit isoform antibody and anti-Na,K-ATPase α1subunit isoform antibody; e) non-immune mouse IgG1 antibody; 0 universalantibody reagent comprising a mixture of anti-rabbit IgG and anti-mouseIgG reagents; g) primary chemical stain; h) general counter-chemicalstain; i) specific cellular organelle stain; or j) a combination of twoor more thereof.
 50. The method of claim 49 comprising: a) providing asample of mammalian tissue; b) immunochemically staining the α3 isoformand α1 isoforms of the α-subunit of Na,K-ATPase present in the sample;c) determining the content of α3 isoform of Na, K-ATPase and the contentof α1 isoform of Na, K-ATPase in the sample; and d) determining theratio of α3 isoform to α1 isoform present in the sample.
 51. The methodof claim 37, wherein the disease or disorder having an etiologyassociated with excessive cell proliferation is selected from the groupconsisting of: 1) autoimmune diseases such as antigen-induced arthritisand allergic encephalomyelitis; 2) chronic inflammatory proliferativediseases including rheumatoid arthritis, systemic-onset juvenile chronicarthritis, osteoporosis, and psoriasis; 3) proliferative diseases of thebreast including fibrocystic disease; 4) proliferative diseases of theprostate including benign prostatic hyperplasia (BPH); 5) proliferativediseases of the eye including proliferative diabetic retinopathy; 6)vascular proliferative diseases including atherosclerosis and coronarystenosis; 7) cancer; and 8) tumor.
 52. The method of claim 51, whereinthe cardiac glycoside is selected from the group consisting ofoleandrin, ouabain, bufalin, digitoxin, digoxin, cinobufatalin,cinobufagin, and resibufogenin.
 53. A method of determining whether ornot a subject having a disease or disorder having an etiology associatedwith excessive cell proliferation should be treated with a cardiacglycoside, the method comprising: obtaining a sample of diseased tissuefrom the subject, the disease having an etiology associated withexcessive cell proliferation, and the sample comprising one or moreisoforms of the α-subunit of Na, K-ATPase; determining the ratio of α3isoform to α1 isoform of the α-subunit of Na, K-ATPase in the sample,wherein the step of determining said ratio comprises quantifying thelevel of expression of each of the α3 subunit isoform of Na, K ATPaseand the α1 subunit isoform of Na, K-ATPase in the in vitro sample orbiopsy sample, and calculating the ratio thereof; or the step ofdetermining the ratio comprises determining the amount of each of the α3subunit isoform of Na, K-ATPase relative to amount of the α1 subunitisoform of Na, K-ATPase in the in vitro sample, and calculating theratio thereof; and if said ratio is ≧0.3, the subject should be treatedwith said cardiac glycoside by administration of a compositioncomprising said cardiac glycoside to the subject according to aprescribed dosing regimen, or if said ratio is <0.3, the subject shouldnot be treated with said cardiac glycoside for treatment of said diseaseor disorder having an etiology associated with excessive cellproliferation.
 54. The method of claim 53, wherein if the ratio is ≧0.5,≧1, or ≧10, indicating that the subject should be treated with cardiacglycoside by administration of a composition comprising cardiacglycoside to the subject according to a prescribed dosing regimen.